Novel cellulases, the genes encoding them and uses thereof

ABSTRACT

Genes encoding novel cellulases, and a gene encoding a protein that facilitates the action of such novel cellulases, the novel cellulases and a protein that facilitates the action of such cellulases, and enzyme preparations containing such proteins are described. The native hosts and the culture medium of said hosts containing said novel cellulases are also disclosed. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention is related to genes encoding novel neutralcellulases and compositions containing the novel neutral cellulases.These compositions are especially useful in the textile, detergent andpulp and paper industries.

[0003] 2. Related Art

[0004] Cellulose is a linear polysaccharide of glucose residuesconnected by β-1,4 linkages. In nature, cellulose is usually associatedwith lignin together with hemicelluloses such as xylans andglucomannans. The practical use of cellulases has been hampered by thenature of the known cellulases, which are often mixtures of cellulaseshaving a variety of activities and substrate specificities. For thatreason, it is desirable to identify sources from which cellulases havingonly the desired activities may be obtained.

[0005] A wide variety of cellulases are known in the art, most of whichare acid cellulases. However, some neutral and alkaline cellulases havealso been identified. Celluzyme® is a commercially-available cellulasepreparation from Humicola insolens (Novo Nordisk, A/S). GB 2,075,028 andEP 406,314 describe the use of a Humicola insolens cellulase as anenzymatic additive in a wash detergent to reduce the harshness(stiffness) of cotton-containing fabrics. The cloning of a cellulasecontaining endoglucanase activity from Humicola insolens is described inWO 93/11249 and EP 531,372. EP 510,091 describes a cellulase fromBacillus spp. NCIMB 40250 that is useful in detergent compositions. EP220,016 describes cellulases that are useful as clarification agents forcolored fabrics. WO 94/07998 describes modified cellulases that possessan improved alkaline activity. WO 95/02675 describes detergentcompositions that contain two different cellulases: a first cellulasethat is catalytically amenable to particulate soil removal, and a secondcellulase that is catalytically amenable to color clarification. WO92/18599 describes a detergent preparation that contains both acellulase and a protease. Cellulases have also been used industrially asan aid for the removal of printing paste thickener and excess dye aftertextile printing (EP 576,526).

[0006] EP 383 828 describes granular detergent compositions, whichcontain surface-active agent, a fabric-softening clay material, andcellulase granulates containing calcium carbonate. U.S. Pat. No.5,433,750 describes detergent compositions containing a surface activeagent, a builder system, a softening clay, a clay flocculating agent anda high activity cellulase, preferably Humicola insolens cellulase. U.S.Pat. No. 5,520,838 describes granular detergent compositions, comprisingsurface-active agent, a builder and a cellulase, preferably a Humicolainsolens cellulase, said compositions being in a compact form, having arelatively high density and containing a low amount of inorganic fillersalt.

[0007] Cellulase enzymes are used in a wide variety of industries inaddition to the textile industry. For example, cellulases are usedindustrially for the deinking of newspapers and magazines (EP 521,999),for improving the drainage of pulp (WO 91/14822, WO 91/17243), and as atreatment for animal feed.

[0008] The unique properties of each cellulase make some more suitablefor certain purposes than others. While the enzymes differ in a numberof ways, one of the most important difference is pH optimum. Neutralcellulases have useful cellulase activity in the pH range 6-8, alkalinecellulases have useful cellulase activity in the pH range 7.5-10. Acidcellulases are active in the range of pH 4.5-6, but have littlecellulase activity at higher pH values.

[0009] Neutral and acid cellulases are especially useful in the textileindustry Klahorst, S. et al., Textile Chemist and Colorist 26:13-18,1994; Nilsson, T. E., Aachen Textile Conference, DWI Reports 114:85-88(1995); Videbaek, T. et al., ITB Dyeing/Printing/Finishing, January1994, pp. 25-29; Klahorst, S. et al., AATCC Int. Conf. & Exhibit, Oct.4-7, 1992, p. 243, Atlanta, Ga.; Kochavi, D. et al., Am. DyestuffResporter, September 1990, pp. 26-28; Tyndall, R. Michael, TextileChemist and Colorist 24:23 (1992); Lange, N. K., in Proc. Second TRICELSymp. on Trichoderma reesei Cellulases and Other Hydrolases, Espoo,Finland, 1993, ed. P. Suominen et al., Foundation for Biotechnical andIndustrial Fermentation Research vol. 8, 1993, pp. 263-272. When used totreat fabric, cellulases attack the chains of cellulose molecules thatform the cotton fibers, thereby affecting the characteristics of thefabric.

[0010] Traditionally, in “stonewashing,” pumice stones have been used tochange the characteristics of the fabric. Gradually, cellulases arereplacing pumice stones, which also give the fabric its desired finallook but can cause damage to the machines, garments and sewageprocessing equipment. U.S. Pat. No. 4,832,864, U.S. Pat. No. 4,912,056,U.S. Pat. No. 5,006,126, U.S. Pat. No. 5,122,159, U.S. Pat. No.5,213,581 and EP 307,564 disclose the use of cellulases in biostoning.

[0011] Cellulases are especially useful for stonewashing denim dyed withindigo as the dye mostly stays on the surface of the yarn and does notpenetrate the fibers well. When used to treat cotton fabric, neutralcellulases generally require a longer wash time than do the acidcellulases. However, available neutral cellulases are less aggressive(active) against cotton than acid cellulases, and are reported not tocompromise the strength of the fabric as readily as acid cellulases.Neutral cellulases have a broader pH profile and thus the pH increasethat occurs during biostoning has little effect on the activity of theneutral enzyme.

[0012] The use of acid cellulases is hampered by their tendency topromote backstaining and a weakening of fabrics. In addition, the pHmust be adjusted to to a range suitable for the function of the acidcellulases. Consequently, there is a clear demand for neutral cellulaseenzyme preparations that do not cause backstaining or weakening offabrics.

[0013] While it has become popular to use cellulases in the textileindustry, simply changing the cellulase mixture that is used may producea different finish. These problems have focused increasing attention onthe search for reproducible mixtures of cellulases with desiredproperties. Thus there is a clear demand especially in the textile anddetergent industry for novel cellulases active at neutral and alkalinepH values, not compromising the strength of fabrics, with good cleaningand/or fabric care and harshness reducing properties.

SUMMARY OF THE INVENTION

[0014] Recognizing the importance of identifying enzymes useful intextile biofinishing and biostoning and in detergent applications, theinventors have screened fungal species for neutral and alkalinecellulases with enzymatic characteristics that would be useful in suchtechnologies.

[0015] These studies have resulted in novel cellulases originating fromthe genera Myceliophthora, Myriococcum, Melanocarpus, Sporotrichum andChaetomium.

[0016] The invention is further directed to the spent culture medium orenzyme preparations prepared from the native hosts producing such novelcellulases.

[0017] The invention is further directed to the use of such culturemedium or the use of such enzyme preparations in the textile anddetergent industry and in the pulp and paper industries.

[0018] These studies have further resulted in the identification ofthree novel cellulases that are especially useful in the textile anddetergent industry. Purified preparations from Melanocarpus sp. orMyriococcum sp. have revealed a 20 kDa cellulase with endoglucanaseactivity (designated herein as “20K-cellulase”), a 50 kDa cellulase(“50K-cellulase”), and a second 50 kDa cellulase (“50K-cellulase B”). Anovel gene product with high homology to the cellulase family, hereincalled “protein-with-CBD” (where CBD means “cellulose binding domain”)was also discovered.

[0019] It is an object of the invention to provide enzyme preparationsthat contain one or more of the novel cellulases of the invention,especially the 20K-cellulase, the 50K-cellulase, the 50K-cellulase Band/or the protein-with-CBD.

[0020] It is a further object of this invention to provide a method forusing such preparations for the finishing of textiles, especially thebiostoning of denim, for the use said preparations in detergentcompositions, and especially methods that use the 20K-cellulase, the50K-cellulase, the 50K-cellulase B and/or the protein-with-CBD.

[0021] The invention is also directed to other neutral and/or alkalinecellulases having one or more of the amino acid sequences as describedherein.

[0022] The invention is further directed to the genes encoding the20K-cellulase, 50K-cellulase, 50K-cellulase B and the protein-with-CBD.

[0023] The invention is further directed to novel expression vectorscomprising such genes and to novel hosts transformed with the vectors,especially hosts that are capable of high levels of expression of theproteins encoded by such genes.

[0024] The invention is further directed to the spent culture medium ofsuch transformed hosts, the culture medium containing the novel20K-cellulase, 50K-cellulase, the 50K-cellulase B and/or theprotein-with-CBD, or enzyme compositions (enzyme preparations)containing one or more of these proteins that have been prepared fromsuch culture media.

[0025] The invention is further directed to the use of such culturemedium or the use of such enzyme preparations in the textile anddetergent industry and in the pulp and paper industries.

BRIEF DESCRIPTION OF THE FIGURES

[0026]FIG. 1 (A and B) show the pH (FIG. 1A) and temperature (FIG. 1B)dependencies of the endoglucanase activities of ALKO4179, CBS 689.95

[0027]FIG. 2 (A and B) show the pH (FIG. 2A) and temperature (FIG. 2B)dependencies of the endoglucanase activities of ALKO4124, CBS 687.95.

[0028]FIG. 3 (A and B) show the pH (FIG. 3A) and temperature (FIG. 3B)dependencies of the endoglucanase activities of ALKO4237, CBS 685.95.

[0029]FIG. 4 (A and B) show the pH (FIG. 4A) and temperature (FIG. 4B)dependencies of the endoglucanase activities of ALKO4265, CBS 730.95.

[0030]FIG. 5 (A and B) show the pH (FIG. 5A) and temperature (FIG. 5B)dependencies of the endoglucanase activities of ALKO4125, CBS 688.95.

[0031]FIG. 6 (A and B) show the wash effect and backstaining (FIG. 6A)and blueness (FIG. 6B) with the neutral cellulases.

[0032]FIG. 7 (A and B) show the wash effect and backstaining (FIG. 7A)and blueness (FIG. 7B) with Ecostone L with gradually increasing enzymedosages. 1× corresponds the enzyme dosage of the neutral cellulases inFIGS. 6A and 6B.

[0033]FIG. 8 shows the purification of 20K-cellulase from Peak II bychromatography on SP-Sepharose™. A sample containing 11.7 g of proteinand 576,000 ECU was applied to a 4.5×31 cm column, which was developedas described in Example 9. Fractions of 15 ml were collected.Endoglucanase activities in the peak at fractions 148-161 areunderestimated because crystallization occurred before the enzyme couldbe sufficiently diluted for assay. Crystalline material from thesefractions contained 486,000 ECU.

[0034]FIG. 9 (A and B) show SDS-PAGE analysis of the 20K-cellulase. Themolecular masses of the standards are shown in kDa.

[0035] A Partially crystalline material precipitated from the activeS-Sepharose™ fractions (lane 1).

[0036] B Fractions from chromatography of the partially crystallinematerial on G50 Sephadex. Fractions shown in lanes 19 and 25 containedno endoglucanase activity. For the other fractions, the amounts ofactivity (in ECU) applied to the gel was as follows: fraction 27, 0.4;29, 2.4 (as 3.0 μg of protein); 30, 2.1; 31, 1.9; 33, 0.46; and 35, 1.1.

[0037]FIG. 10 shows the separation of 50K-cellulase and 50K-cellulase Bfrom Peak III/IV by chromatography on SP-Sepharose™. A sample containing200 mg of protein and 14,800 ECU was applied to the 2.5×11 cm column,which was developed as described in Example 9. Fractions of 6.8 ml werecollected. A minor amount of 50K-cellulase eluted before the NaClgradient, whereas most of the 50K-cellulase eluted at about 50 mM NaCl.50K-cellulase B was found in the major protein peak at about 80 mM NaCl.

[0038]FIG. 11 shows an SDS-PAGE analysis of purified 50K-cellulase (11A)and 50K-cellulase B (11B). Lane numbers indicate the fractions (3.3 ml)eluted from Phenyl-Sepharose. For fractions 36-41, 2.5 μl of eachfraction was applied to the gel. For the other fractions, 2 μl wasapplied. The 50K-cellulase peak was found in fractions 37-38 (11A)(containing 780 and 880 ECU/ml, respectively). The 50K-cellulase B peakwas in fractions 30 and 31 (11B), which contained negligible activity(less than 4 ECU/ml).

[0039]FIG. 12 shows the temperature dependence of the endoglucanaseactivity of 50K-cellulase at pH 7.0 and a reaction time of 60 min.

[0040]FIG. 13 shows the pH dependence of the endoglucanase activity of50K-cellulase at 50° C. (♦) and 70° C. (□).

[0041]FIG. 14 shows a Western analysis using 20K-cellulase antiserum asa probe. Lanes 1, 2 and 3 contain 25 μg of protein from theDEAE-Sepharose peaks I, III and IV, respectively. Lanes 4 and 5 contain2.0 and 0.2 μg of pure 50K-cellulase and lane 6 contains 0.6 μg of pure50K-cellulase B. Lanes 7 and 8 contain about 25 μg protein from thewhole growth medium of ALKO4237 and ALKO4124, respectively.

[0042]FIG. 15 shows the temperature dependence of the endoglucanaseactivity of 20K-cellulase at pH 7 (10 min reaction times).

[0043] FIGS. 16 (A and B) show the pH-dependence of the endoglucanaseactivity of the 20K-cellulase for the reaction time of (a) 10 minutes or(b) 60 minutes.

[0044]FIG. 17 shows amino acid sequence data derived from sequencing the20K-cellulase described in the exemplary material herein. Sequence 429is from the N terminus of the protein: and the other sequences are frominternal tryptic peptides.

[0045]FIG. 18 shows the restriction maps of the Melanocarpus albomycesDNA in plasmids pALK1221, pALK1222 and pALK1223, which carry the20K-cellulase gene.

[0046]FIG. 19 shows the DNA sequence of the 20K-cellulase gene. Thearrow indicates the predicted signal peptidase processing site.

[0047]FIG. 20 shows the restriction maps of the Melanocarpus albomycesDNA in plasmids pALK1233, pALK1234, pALK1226 and pALK1227, which carrythe 50K-cellulase gene.

[0048]FIG. 21 (A and B) show the DNA sequence of the 50K-cellulase gene.The arrow indicates the predicted signal peptidase processing site.

[0049]FIG. 22 shows the restriction maps of the Melanocarpus albomycesDNA in plasmids pALK1229 and pALK1236, which carry the 50K-cellulase Bgene.

[0050]FIG. 23 (A and B) show the DNA sequence of the 50K-cellulase Bgene The arrow indicates the predicted signal peptidase processing site.

[0051]FIG. 24 shows the plasmid map of pTTc01.

[0052]FIG. 25 shows the plasmid map of pMS2.

[0053]FIG. 26 shows the restiction map of the Melanocarpus albomyces DNAin plasmid pALK1230, which carries DNA encoding the protein-with-CBD.The sequence presented in FIG. 27 is marked with an arrow in FIG. 26.

[0054]FIG. 27 shows the DNA sequence of the the protein-with-CBDcellulase gene in pALK1230.

[0055]FIG. 28 shows the plasmid map of pALK1231.

[0056]FIG. 29 shows the plasmid map of pALK1235.

[0057]FIG. 30 shows a Western analysis using 20K-cellulase antiserum asa probe. Lanes 1 and 2 contain about 10 μg protein from the whole growthmedium of transformants ALKO3620/pALK1235/49 and ALKO3620/pALK1235/40.Lane 3 contains about 10 μg protein from the whole growth medium ofALKO3620. Lanes 4 and 5 contain about 10 μg protein from the wholegrowth medium of transformants ALKO3620/pALK1231/16 andALKO3620/pALK1231/14. Lane 6 contains 100 ng of pure 20K-cellulase.

[0058]FIG. 31 shows the plasmid map of pALK1238.

[0059]FIG. 32 shows the plasmid map of pALK1240.

Deposits

[0060] ALKO4179, Myceliophihora thermophila was deposited as CBS 689.95on Oct. 12, 1995, at the Centraalbureau voor Schimmelcultures, P.O. Box273, 3740 AG BAARN.

[0061] ALKO4124, Myriococcum sp. was deposited as CBS 687.95 on Oct. 12,1995, at the Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AGBAARN.

[0062] ALKO4237, Melanocarpus albomyces (=Myriococcumalbomyces=Thielavia albomyces; Guarro et al., 1996, Mycol. Res.100(1):75.) was deposited as CBS 685.95 on Oct. 11, 1995, at theCentraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AG BAARN.

[0063] ALKO4125, Sporotrichum thermophile was deposited as CBS 688.95 onOct. 12, 1995, at the Centraalbureau voor Schimmelcultures, P.O. Box273, 3740 AG BAARN.

[0064] ALKO4265, Chaetomium thermophilum La Touche was deposited as CBS730.95 on Nov. 8, 1995, at the Centraalbureau voor Schimmelcultures,P.O. Box 273, 3740 AG BAARN.

[0065] Plasmid pALK1221 was deposited as DSM 11024 on Jun. 21, 1996 andλ4237/5.1 was deposited as DSM 11012 on Jun. 21, 1996, at the DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1B,D-38124 Braunschweig, Germany. Both contain the 20K-cellulase gene fromMelanocarpus albomyces CBS 685.95.

[0066] Plasmid pALK1227 was deposited as DSM 11025 on Jun. 21, 1996 andλ4237/35 was deposited as DSM 11014 on Jun. 21, 1996, at the DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1B,D-38124 Braunschweig, Germany. Both contain the 50K-cellulase gene fromMelanocarpus albomyces CBS 685.95.

[0067] Plasmid pALK1229 was deposited as DSM 11026 on Jun. 21, 1996 andλ4237/3 was deposited as DSM 11011 on Jun. 21, 1996, and λ4237/18 wasdeposited as DSM 11013 on Jun. 21, 1996, at the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1B, D-38124Braunschweig, Germany. pALK1229 contains DNA coding for the50K-cellulase B, λ4237/3 and λ4237/18 contain the 50K-cellulase B genefrom Melanocarpus albomyces CBS 685.95.

[0068] Plasmid pALK1230 was deposited as DSM 11027 on Jun. 21, 1996 atthe Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Mascheroder Weg 1B, D-38124 Braunschweig, Germany. pALK1230 contains theprotein-with-CBD gene from Melanocarpus albomyces CBS 685.95.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] In the description that follows, a number of terms used intextile industry technology are extensively utilized. In order toprovide a clear and consistent understanding of the specification andclaims, including the scope to be given such terms, the followingdefinitions are provided.

[0070] Biostoning. “Biostoning” of fabric or garment means the use ofenzymes in place of, or in addition to, the use of pumice stones for thetreatment of fabric or garment, especially denim.

[0071] Biofinishing. “Biofinishing” refers to the use of enzymes in acontrolled hydrolysis of cellulosic fibers in order to modify the fabricor yarn surface in a manner that prevents permanently pilling, improvesfabric handle like softness and smoothness, clears the surface structureby reducing fuzzing, which results in clarification of colours, improvesthe drapability of the fabric, improves moisture absorbability and whichmay improve also the dyeability.

[0072] Backstaining. Released dye has a tendency to redeposit on thesurface of the fabric fibers. This effect is termed “backstaining.”

[0073] Detergent. By “detergent” is meant a cleansing agent that cancontain surface active agents (anionic, non-ionic, cationic andampholytic surfactants), builders and other optional incredients such asantiredeposition and soil suspension agents, optical brighteners,bleaching agents, dyes and pigments and hydrolases. Suitable listing ofthe contents of detergents is given in U.S. Pat. No. 5,433,750, asuitable list of surfactants is given in U.S. Pat. No. 3,664,961.

[0074] Enzyme preparation. By “enzyme preparation” is meant acomposition containing enzymes. Preferably, the enzymes have beenextracted from (either partially or completely purified from) a microbeor the medium used to grow such microbe. “Extracted from” means that thedesired enzymes are separated from the cellular mass. This can beperformed by any method that achieves this goal, including breakingcells and also simply removing the culture medium from spent cells.Therefore, the term “enzyme preparation” includes compositionscontaining medium previously used to culture a desired microbe(s) andany enzymes that have been released from the microbial cells into suchmedium during the culture or downstream processing steps.

[0075] By a host that is “substantially incapable” of synthesizing oneor more enzymes is meant a host in which the activity of one or more ofthe listed enzymes is depressed, deficient, or absent when compared tothe wild-type.

[0076] By an amino acid sequence that is an “equivalent” of a specificamino acid sequence is meant an amino acid sequence that is notidentical to the specific amino acid sequence, but rather contains atleast some amino acid changes (deletions, substitutions, inversions,insertions, etc) that do not essentially affect the biological activityof the protein as compared to a similar activity of the specific aminoacid sequence, when used for a desired purpose. The biological activityof a cellulase, is its catalytic activity, and/or its ability to bind tocellulosic material. The biological activity of the 50K-cellulase Bfurther includes its ability to act synergistically with the cellulases.Preferably, an “equivalent” amino acid sequence contains at least80%-99% identity at the amino acid level to the specific amino acidsequence, most preferably at least 90% and in an especially highlypreferable embodiment, at least 95% identify, at the amino acid level.

[0077] Cloning vehicle. A cloning vehicle is a plasmid or phage DNA orother DNA sequence (such as a linear DNA) that provides an appropriatenucleic acid carrier environment for the transfer of a gene of interestinto a host cell. The cloning vehicles of the invention may be designedto replicate autonomously in prokaryotic and eukaryotic hosts. In fungalhosts such as Trichoderma, the cloning vehicles generally do notautonomously replicate and instead, merely provide a vehicle for thetransport of the gene of interest into the Trichoderma host forsubsequent insertion into the Trichoderma genome. The cloning vehiclemay be further characterized by one or a small number of endonucleaserecognition sites at which such DNA sequences may be cut in adeterminable fashion without loss of an essential biological function ofthe vehicle, and into which DNA may be spliced in order to bring aboutreplication and cloning of such DNA. The cloning vehicle may furthercontain a marker suitable for use in the identification of cellstransformed with the cloning vehicle. Markers, for example, areantibiotic resistance. Alternatively, such markers may be provided on acloning vehicle which is separate from that supplying the gene ofinterest. The word “vector” is sometimes used for “cloning vehicle.”

[0078] Expression vehicle. An expression vehicle is a cloning vehicle orvector similar to a cloning vehicle but which is capable of expressing agene of interest, after transformation into a desired host. When afungal host is used, the gene of interest is preferably provided to afungal host as part of a cloning or expression vehicle that integratesinto the fungal chromosome, or allows the gene of interest to integrateinto the host chromosome. Sequences that are part of the cloning vehicleor expression vehicle may also be integrated with the gene of interestduring the integration process. In T. reesei, sites of integration towhich the gene of interest can be directed include the cbh and/or theegl loci. Most preferably, the gene of interest is directed to replaceone or more genes encoding undesirable characteristics.

[0079] The gene of interest is also preferably placed under the controlof (i.e., operably linked to) certain control sequences such as promotersequences provided by the vector (which integrate with the gene ofinterest). Alternatively, the control sequences can be those at theinsertion site.

[0080] The expression control sequences of an expression vector willvary depending on whether the vector is designed to express a certaingene in a prokaryotic or in a eukaryotic host (for example, a shuttlevector may provide a gene for selection in bacterial hosts). Expressioncontrol sequences can contain transcriptional regulatory elements suchas, promoters, enhancer elements, and transcriptional terminationsequences, and/or translational regulatory elements, such as, forexample, translational initiation and termination sites.

[0081] According to the invention, there are provided neutral andalkaline cellulases, and methods for producing such useful neutral andalkaline cellulases, that are desirable for the treatment of textilematerials.

[0082] The native hosts that produce the proteins of the invention are:

[0083] 1) ALKO4179, Myceliophthora thermophila; deposited as CBS 689.95at the Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AGBAARN.

[0084] 2) ALKO4124, Myriococcum sp.; deposited as CBS 687.95;

[0085] 3) ALKO4237, Melanocarpus albomyces, deposited as CBS 685.95;

[0086] 4) ALKO4125, Sporotrichum thermophila, deposited as CBS 688.95;and

[0087] 5) ALKO4265, Chaetomium thermophilum La Touche, deposited as CBS730.95

[0088] One specific preferred embodiment of the invention is the spentculture medium of the native hosts or enzyme preparations prepared fromthe culture medium.

[0089] In specific preferred embodiments of the invention, the purified20K-cellulase, 50K-cellulase, 50K-cellulase B and/or protein-with-CBD isprovided. These proteins can be obtained for example from Melanocarpussp. or Myriococcum sp. as described herein, and especially in Example 9.

[0090] Amino acid sequence data have been generated from the cellulasesdescribed herein. Accordingly, the invention is also directed to neutralor alkaline cellulases containing one or more of the amino acidsequences shown herein. Thus, the invention is intended to be directedto any neutral or alkaline cellulase that is a functional equivalent ofthe 20K-cellulase, the 50K-cellulase, the 50K-cellulase B and/orprotein-with-CBD and having one or more of the amino acid sequencesdescribed herein, or substantially the same sequence. Such neutral oralkaline cellulases can be derived from other strains of the samespecies or from divergent organisms.

[0091] In further preferred embodiments, the 20K-cellulase is providedwith the material from separate peaks formed during the exemplifiedpurification procedures (e.g., DEAE-Sepharose Pools I, III, or IV inTable VIII herein). In still further embodiments, other proteins in theMelanocarpus albomyces ALKO 4237 medium may be used, either alone or incombination with other such proteins.

[0092] In further preferred embodiments, the 50K-cellulase is providedwith the material from separate peaks formed during the exemplifiedpurification procedures. In still further embodiments, other proteins inthe ALKO 4237 medium may be used, either alone or in combination withother such proteins.

[0093] In further preferred embodiments, the 50K-cellulase B is providedwith the material from separate peaks formed during the exemplifiedpurification procedures. In still further embodiments, other proteins inthe ALKO 4237 medium may be used, either alone or in combination withother such proteins.

[0094] As described herein, ALKO 4265, Chaetomium thermophilum LaTouche, deposited as CBS 730.95, is used herein as an example of aneutral cellulase that is not preferred in biostoning method of theinvention because it causes backstaining. However, there is evidencethat it is useful in other applications (e.g. in detergents).

[0095] The process for genetically engineering the hosts of theinvention is facilitated through the cloning of genetic sequences thatencode the desired protein and through the expression of such geneticsequences. As used herein the term “genetic sequences” is intended torefer to a nucleic acid molecule (preferably DNA). Genetic sequencesthat encode the desired protein are derived from a variety of sources.These sources include genomic DNA, cDNA, synthetic DNA and combinationsthereof. Vector systems may be used to produce hosts for the productionof the enzyme preparations of the invention. Such vector construction(a) may further provide a separate vector construction (b) which encodesat least one desired gene to be integrated to the genome of the host and(c) a selectable marker coupled to (a) or (b). Alternatively, a separatevector may be used for the marker.

[0096] A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains expression control sequenceswhich contain transcriptional regulatory information and such sequencesare “operably linked” to the nucleotide sequence which encodes thepolypeptide.

[0097] An operable linkage is a linkage in which a sequence is connectedto a regulatory sequence (or sequences) in such a way as to placeexpression of the sequence under the influence or control of theregulatory sequence. Two DNA sequences (such as a protein encodingsequence and a promoter region sequence linked to the 5′ end of theencoding sequence) are said to be operably linked if induction ofpromoter function results in the transcription of the protein encodingsequence mRNA and if the nature of the linkage between the two DNAsequences does not (1) result in the introduction of a frame-shiftmutation, (2) interfere with the ability of the expression regulatorysequences to direct the expression of the mRNA, antisense RNA, orprotein, or (3) interfere with the ability of the template to betranscribed by the promoter region sequence. Thus, a promoter regionwould be operably linked to a DNA sequence if the promoter were capableof effecting transcription of that DNA sequence.

[0098] The precise nature of the regulatory regions needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribing and 5′ non-translating(non-coding) sequences involved with initiation of transcription andtranslation respectively. Expression of the protein in the transformedhosts requires the use of regulatory regions functional in such hosts. Awide variety of transcriptional and translational regulatory sequencescan be employed. In eukaryotes, where transcription is not linked totranslation, such control regions may or may not provide an initiatormethionine (AUG) codon, depending on whether the cloned sequencecontains such a methionine. Such regions will, in general, include apromoter region sufficient to direct the initiation of RNA synthesis inthe host cell.

[0099] As is widely known, translation of eukaryotic mRNA is initiatedat the codon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes the protein, or a functional derivativethereof, does not contain any intervening codons which are capable ofencoding a methionine. The presence of such codons results either in aformation of a fusion protein (if the AUG codon is in the same readingframe as the protein encoding DNA sequence) or a frame-shift mutation(if the AUG codon is not in the same reading frame as the proteinencoding sequence).

[0100] In a preferred embodiment, a desired protein is secreted into thesurrounding medium due to the presence of a secretion signal sequence.If a desired protein does not possess its own signal sequence, or ifsuch signal sequence does not function well in the host, then theprotein's coding sequence may be operably linked to a signal sequencehomologous or heterologous to the host. The desired coding sequence maybe linked to any signal sequence which will allow secretion of theprotein from the host. Such signal sequences may be designed with orwithout specific protease sites such that the signal peptide sequence isamenable to subsequent removal. Alternatively, a host that leaks theprotein into the medium may be used, for example a host with a mutationin its membrane.

[0101] If desired, the non-transcribed and/or non-translated regions 3′to the sequence coding for a protein can be obtained by theabove-described cloning methods. The 3′-non-transcribed region may beretained for its transcriptional termination regulatory sequenceelements; the 3-non-translated region may be retained for itstranslational termination regulatory sequence elements, or for thoseelements which direct polyadenylation in eukaryotic cells.

[0102] The vectors of the invention may further comprise other operablylinked regulatory elements such as enhancer sequences.

[0103] In a preferred embodiment, genetically stable transformants areconstructed whereby a desired protein's DNA is integrated into the hostchromosome. The coding sequence for the desired protein may be from anysource. Such integration may occur de novo within the cell or, in a mostpreferred embodiment, be assisted by transformation with a vector whichfunctionally inserts itself into the host chromosome, for example, DNAelements which promote integration of DNA sequences in chromosomes.

[0104] Cells that have stably integrated the introduced DNA into theirchromosomes are selected by also introducing one or more markers whichallow for selection of host cells which contain the expression vector inthe chromosome, for example the marker may provide biocide resistance,e.g., resistance to antibiotics, or heavy metals, such as copper, or thelike. The selectable marker gene can either be directly linked to theDNA gene sequences to be expressed, or introduced into the same cell byco-transformation.

[0105] Factors of importance in selecting a particular plasmid or viralvector include: the ease with which recipient cells that contain thevector may be recognized and selected from those recipient cells whichdo not contain the vector; the number of copies of the vector which aredesired in a particular host; and whether it is desirable to be able to“shuttle” the vector between host cells of different species.

[0106] Once the vector or DNA sequence containing the construct(s) isprepared for expression, the DNA construct(s) is introduced into anappropriate host cell by any of a variety of suitable means, includingtransformation as described above. After the introduction of the vector,recipient cells are grown in a selective medium, which selects for thegrowth of transformed cells. Expression of the cloned gene sequence(s)results in the production of the desired protein, or in the productionof a fragment of this protein. This expression can take place in acontinuous manner in the transformed cells, or in a controlled manner.

[0107] Accordingly, the protein encoding sequences described herein maybe operably linked to any desired vector and transformed into a selectedhost, so as to provide for expression of such proteins in that host.

[0108] The subject matter of the invention are also nucleic acidmolecules coding for proteins having the biological activity of acellulase and that hybridize to any of the nucleic acid moleculesdescribed above or which are defined in the following:

[0109] A nucleic acid molecule encoding a polypeptide having theenzymatic activity of a cellulase, selected from the group consistingof:

[0110] (a) nucleic acid molecules encoding a polypeptide comprising theamino acid sequence as depicted in FIG. 19 or 21;

[0111] (b) nucleic acid molecules encoding a polypeptide comprising theamino acid sequence as depicted in FIG. 23 or 27;

[0112] (c) nucleic acid molecules comprising the coding sequence of thenucleotide sequence as depicted in FIG. 19 or 21;

[0113] (d) nucleic acid molecules comprising the coding sequence of thenucleotide sequence as depicted in FIG. 23 or 27;

[0114] (e) nucleic acid molecules encoding a polypeptide comprising theamino acid sequence encoded by the DNA insert contained in DSM 11024,DSM 11012, DSM 11025 or DSM 11014;

[0115] (f) nucleic acid molecules encoding a polypeptide comprising theamino acid sequence encoded by the DNA insert contained in DSM 11026,DSM 11011, DSM 11013 or DSM 11027;

[0116] (g) nucleic acid molecules comprising the coding sequence of theDNA insert contained in DSM 11024, DSM 11012, DSM 11025 or DSM 11014;

[0117] (h) nucleic acid molecules comprising the coding sequence of theDNA insert contained in DSM11026, DSM 11011, DSM 11013 or DSM 11027;

[0118] (i) nucleic acid molecules hybridizing to a molecule of any oneof (a), (c), (e) or (g); and

[0119] (j) nucleic acid molecules the coding sequence of which differsfrom the coding sequence of a nucleic acid molecule of any one of (a) to

[0120] (i) due to the degeneracy of the genetic code.

[0121] (k) nucleic acid molecules encoding a polypeptide havingcellulase activity and having an amino acid sequence which shows atleast 80% identity to a sequence as depicted in FIGS. 19, 21, 23 or 27.

[0122] The term “hybridization” in this context means hybridizationunder conventional hybridization conditions, preferably under stringentconditions such as described by, e.g. Sambrook et al. (1989, MolecularCloning, A Laboratory Manual 2nd Edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). These nucleic acid molecules thathybridize to the nucleic acid molecules according to the presentinvention in principle can be derived from any organism possessing suchnucleic acid molecules. Preferably, they are derived from fungi, namelyfrom those of the genera Melanocarpus, Myriococcum, Sporotrichum,Myceliophthora and Chaetomium. Nucleic acid molecules hybridizing to thenucleic acid molecules of the present invention can be isolated, e.g.,from genomic libraries or cDNA libraries of various organisms, namelyfungi.

[0123] Such nucleic acid molecules can be identified and isolated byusing the nucleic acid molecules of the present invention or fragmentsof these molecules or the reverse complements of these molecules, e.g.by hybridization according to standard techniques (see Sambrook et al.(1989)).

[0124] As hybridization probe, e.g. nucleic acid molecules can be usedthat have exactly or substantially the same nucleotide sequenceindicated in the Figures or fragments of said sequence. The fragmentsused as hybridization probes can also be synthetic fragments obtained byconventional synthesis techniques and the sequence of which issubstantially identical to that of the nucleic acid molecules accordingto the invention. Once genes hybridizing to the nucleic acid moleculesof the invention have been identified and isolated it is necessary todetermine the sequence and to analyze the properties of the proteinscoded for by said sequence.

[0125] The term “hybridizing DNA molecule” includes fragments,derivatives and allelic variants of the above-described nucleic acidmolecules that code for the above-described protein or a biologicallyactive fragment thereof. Fragments are understood to be parts of nucleicacid molecules long enough to code for the described protein or abiologically active fragment thereof. The term “derivative” means inthis context that the nucleotide sequences of these molecules differfrom the sequences of the above-described nucleic acid molecules in oneor more positions and are highly homologous to said sequence. Homologyis understood to refer to a sequence identity of at least 40%,particularly an identity of at least 0.60%, preferably more than 80% andstill more preferably more than 90%. The deviations from the nucleicacid molecules described above can be the result of deletion,substitution, insertion, addition or combination.

[0126] Homology furthermore means that the respective nucleotidesequences or encoded proteins are functionally and/or structurallyequivalent. The nucleic acid molecules that are homologous to thenucleic acid molecules described above and that are derivatives of saidnucleic acid molecules are regularly variations of said molecules whichrepresent modifications having the same biological function. They may benaturally occurring variations, such as sequences of other organisms ormutations. These mutations may occur naturally or may be achieved byspecific mutagenesis. Furthermore, these variations may be syntheticallyproduced sequences. The allelic variants may be naturally occurringvariants as well as synthetically produced or genetically engineeredvariants.

[0127] The proteins encoded by the various variants of the nucleic acidmolecules of the invention share specific common characteristics, suchas enzymatic activity, molecular weight, immunological reactivity,conformation, etc., as well as physical properties, such aselectrophoretic mobility, chromatographic behaviour, sedimentationcoefficients, solubility, spectroscopic properties, stability, pHoptimum, temperature optimum, etc. Enzymatic activity of the cellulasecan be detected e.g. as described on page 11 and in Examples 1 and 25.

[0128] The present invention furthermore relates to nucleic acidmolecules the sequences of which differ from the sequences of theabove-identified molecules due to degeneracy of the genetic code, andwhich code for a protein having the biological activity of a cellulase.

[0129] The nucleic acid molecules of the invention are preferably RNA orDNA molecules, most preferably genomic DNA or cDNA.

[0130] The present invention also relates to antibodies whichspecifically recognize one of the above-described proteins according tothe invention as well as to antibody fragments which have this property.These antibodies may be monoclonal or polyclonal. Methods for theirproduction are well known in the art and are described in detail, forexample, in Harlow and Lane “Antibodies, A Laboratory Manual”, CSHPress, Cold Spring Harbor Laboratory (1988).

[0131] Furthermore, the present invention relates to oligonucleotideswhich specifically hybridize with a nucleic acid molecule according tothe invention or with the complementary strand of such a nucleic acidmolecule. In this respect the term “specifically hybridize” means thatsuch an oligonucleotide hybridizes under stringent hybridizationconditions specifically to a nucleic acid molecule of the invention anddoes not show under such conditions cross-hybridization with sequencescoding for other polypeptides. Preferably such oligonucleotides have alength of at least 10 nucleotides, more preferably of at least 15nucleotides and most preferably of at least 30 nucleotides. They arepreferably no longer than 100 nucleotides, more preferably no longerthan 80 nucleotides and most preferably no longer than 60 nucleotides.In order to ensure that they specifically hybridize to a nucleic acidmolecule of the present invention such oligonucleotides show over theirtotal length an identity of at least 80%, preferably of at least 95% andmost preferably of at least 99% with a corresponding nucleotide sequenceof a nucleic acid molecule of the present invention. Theseoligonucleotides may be used, e.g., as probes for screening forsequences encoding cellulases in genomic or cDNA libraries or as PCRprimers.

[0132] The protein encoding sequences described herein may be fused inframe to other sequences so as to construct DNA encoding a fusionprotein. For example, a recombinant vector encoding a 50K-cellulase, a20K-cellulase, a 50K-cellulase B or the protein-with-CBD gene can beprepared as above, except that the protein encoding sequence is fusedwith the sequence of a T. reesei cellulase, hemicellulase or mannanase,or at least one functional domain of such cellulase, hemicellulase, ormannanase as described in U.S. Pat. No. 5,298,405, WO 93/24622 and inGenBank submission L25310, each incorporated herein by reference.Especially, the cellulase, hemicellulase, or mannanase is selected fromthe group consisting of CBHI, CBHII, EGI, EGII, XYLI, XYLII and MANI, ora domain thereof, such as the secretion signal or the core sequence.Mannanase has the same domain structure as that of the cellulases: acore domain, containing the active site, a hinge domain containing aserine-threonine rich region, and a tail, containing the binding domain.

[0133] Fusion peptides can be constructed that contain a mannanase orcellobiohydrolase or endoglucanase or xylanase core domain or the coreand the hinge domains from the same, fused to the desired proteinencoding sequence of the invention. The result is a protein thatcontains mannanase or cellobiohydrolase or endoglucanase or xylanasecore or core and hinge regions, and a 50K-cellulase, 20K-cellulase,50K-cellulase B or the protein-with-CBD sequence. The fusion proteincontains both the mannanase or cellobiohydrolase or endoglucanase orxylanase, and the 50K-cellulase, 20K-cellulase, 50K-cellulase B or theprotein-with-CBD activities of the various domains as provided in thefusion construct.

[0134] Fusion proteins can also be constructed such that the mannanaseor cellobiohydrolase or endoglucanase or xylanase tail or a desiredfragment thereof, is included, placed before the 50K-cellulase,20K-cellulase, 50K-cellulase B or the protein-with-CBD sequence,especially so as to allow use of a nonspecific protease site in the tailas a protease site for the recovery of the 50K-cellulase, 20K-cellulase,50K-cellulase B or the protein-with-CBD sequence from the expressedfusion protein. Alternatively, fusion proteins can be constructed thatprovide for a protease site in a linker that is placed before the50K-cellulase, 20K-cellulase, 50K-cellulase B or the protein-with-CBDsequence, with or without tail sequences.

[0135] New properties for the 20K- and 50K-cellulases and for the50K-cellulase B can be created by fusing domains, such as a cellulosebinding domain (CBD), preferably with its linker, to the proteins of theinvention. Preferably, such CBD's and linkers are the corresponding CBDand linker domains of a Trichoderma cellulase, mannanase or of theMelanocarpus albomyces protein-with-CBD.

[0136] The invention provides methods for producing enzyme preparationsthat are partially or completely deficient in an undesirablecellulolytic activity (that is, in the ability to degrade cellulose) andenriched in the 50K-cellulase, 20K-cellulase, 50K-cellulase B or theprotein-with-CBD protein, as desired for the textile or detergentindustry or for pulp and paper processing. By “deficient in cellulolyticactivity” is meant a reduced, lowered, or repressed capacity to degradecellulose to smaller oligosaccharides. Such cellulolytic activitydeficient preparations, and the making of same by recombinant DNAmethods, are described in U.S. Pat. No. 5,298,405, incorporated hereinby reference. Preferably, the preparation is deficient in EG activities,and/or CBHI activity.

[0137] As described herein, the 50K-cellulase, 20K-cellulase,50K-cellulase B or the protein-with-CBD may be provided directly by thehosts of the invention. Alternatively, spent medium from the growth ofthe hosts, or purified 50K-cellulase, 20K-cellulase, 50K-cellulase B orthe protein-with-CBD therefrom, can be used. Further, if desiredactivities are present in more than one recombinant host, suchpreparations may be isolated from the appropriate hosts and combinedprior to use in the method of the invention.

[0138] To obtain the enzyme preparations of the invention, the native orrecombinant hosts described above having the desired properties (thatis, hosts capable of expressing economically feasible quantities of thedesired 50K-cellulase, 20K-cellulase, 50K-cellulase B orprotein-with-CBD, and optionally, those that are substantially incapableof expressing one or more other, undesired cellulase enzymes) arecultivated under suitable conditions, the desired enzymes are secretedfrom the hosts into the culture medium, and the enzyme preparation isrecovered from said culture medium by methods known in the art.

[0139] The enzyme preparations of the invention can be produced bycultivating the recombinant hosts or native strains in a fermentor on asuitable growth medium (such as, for example, shown in Example 1 or inExample 30).

[0140] The enzyme preparation can be the culture medium with or withoutthe native or transformed host cells, or is recovered from the same bythe application of methods well known in the art. However, because the50K-cellulase, 20K-cellulase or 50K-cellulase B are secreted into theculture media and display activity in the ambient conditions of thecellulolytic liquor, it is an advantage of the invention that the enzymepreparations of the invention may be utilized directly from the culturemedium with no further purification. If desired, such preparations maybe lyophilized or the enzymatic activity otherwise concentrated and/orstabilized for storage. The enzyme preparations of the invention arevery economical to provide and use because (1) the enzymes may be usedin a crude form; isolation of a specific enzyme from the culture mediumis unnecessary and (2) because the enzymes are secreted into the culturemedium, only the culture medium need be recovered to obtain the desiredenzyme preparation; there is no need to extract an enzyme from thehosts. Preferably the host for such production is Trichoderma, andespecially T reesei.

[0141] The enzyme preparations of the invention may be provided as aliquid or as a solid, for example, in a dried powder or granular orliquid form, especially nondusting granules, or a stabilized liquid, orthe enzyme preparation may be otherwise concentrated or stabilized forstorage or use. It is envisioned that enzyme preparations containing oneor more of the neutral cellulases of the invention can be furtherenriched or made partially or completely deficient in specific enzymaticactivities, so as to satisfy the requirements of a specific utility invarious applications e.g. in the textile industry. A mixture of enzymeactivities secreted by a host and especially a fungus, can be chosen tobe advantageous in a particular industrial application, for examplebiostoning.

[0142] The enzyme preparations of the invention can be adjusted tosatisfy the requirements of specific needs in various applications inthe textile, detergent or the pulp and paper industry.

[0143] Blends may be prepared with other macromolecules that are not allsecreted from the same host (for example, other enzymes such asendoglucanases, proteases, lipases, peroxidases, oxidases or amylases)or chemicals that may enhance the performance, stability, or bufferingof the desired enzyme preparation. Non-dusting granules may be coated.Liquid enzyme preparations can be stabilized by adding a polyol such aspropylene glycol, a sugar or sugar alcohol, lactic acid or boric acid,according to established methods. Liquid detergents generally contain upto 90% water and 0-20% organic solvent. Protected forms of the enzymesof the invention may be prepared as described in EP 238,216.

[0144] The enzyme preparations of the invention can contain a surfactantwhich can be anionic, non-ionic, cationic, amphoteric or a mixture ofthese types, especially when used as a detergent composition. Usefuldetergent compositions are described e.g. in WO 94/07998, U.S. Pat. No.5,443,750 and U.S. Pat. No. 3,664,961.

[0145] If required, a desired enzyme may be further purified inaccordance with conventional conditions, such as extraction,precipitation, chromatography, affinity chromatography, electrophoresis,or the like.

[0146] The enzyme preparations of this invention are especially usefulin textile industry preferably in biostoning and in biofinishing or indetergent industry. Other useful areas are in pulp and paper industry.

[0147] Non-enzymatic stonewashing has three steps: desizing, abrasionand aftertreatment. The first step, desizing, involves the removal ofthe starch coating, or that of its derivatives, by amylase. The secondstep, abrasion, when performed without cellulase, is generally performedby washing the denim with pumice stones, and, when lightening isdesired, bleach. The abrasive effect is the result not only of theeffect of the stones but also the rubbing together of the denim fabric.Abrasion is generally followed by the third step, a washing step toremove excess dye, during which softeners or optical brighteners can beadded.

[0148] In enzymatic stonewashing, or biostoning, abrasion with pumicestones is completely or partially eliminated and cellulase is added tofacilitate the abrasion of indigo dye from the fiber surface. After thistreatment, the cellulase is removed with a detergent wash to ensure thatthe mechanical strength of the fiber is not further compromised by thecontinued presence of the enzyme. Treatment with a cellulase(s) cancompletely replace treatment with pumice stones (for example, 1 kgcommercial enzyme per 100 kg stones). However, cellulase treatment canbe combined with pumice stone treatment when it is desired to produce aheavily abraded finish. A peach skin effect in which a fine protrudinghair-like covering is created is also achieved by a wash combining aneutral cellulase with pumice stones. The cellulases of this inventionare useful especially to minimize backstaining and enhance lightening(abrasion) in biostoning.

[0149] Biostoning is preferably performed from about pH 4.5-9.5, andmost preferably between pH 6.0-8.5. The temperature of the reaction canrange from about 40-80° C., preferably between 50-70° C., and mostpreferably between 50-60° C. The liquid ratio (the ratio of the volumeof liquid per weight of fabric) may range from about 2:1-20:1,preferably 4:1-10:1, and most preferably 4:1-7:1. The enzyme dosage canrange from about 25-1500 nkat/g fabric, preferably 50-500 nkat/g fabricand most preferably 75-300 nkat/g fabric.

[0150] The cellulases of the invention are useful in the textileindustry for biofinishing of fabrics or garments e.g. depilling,defuzzing, colour clarification, harshness reduction, the creation ofdifferent finishes (for example, a ‘peach skin,’ ‘worn out,’ ‘sandwashed,’ or ‘antique look’ effect) and biofinishing of yarn (for examplereduction of hairiness, improvement of smoothness). The cellulases ofthis invention can be used in biofinishing in acidic and in neutralconditions.

[0151] The cellulases of this invention are useful in detergentcompositions to improve the textile cleaning effect e.g. soil removal,to improve the fabric-care properties by reducing the harshness of thetextiles, the cellulases having also defuzzing and colour clarificationand restoring effects.

[0152] The textile material that is treated with the enzyme preparationsof the invention may be manufactured of natural cellulose containingfibers or manmade cellulose containing fibers or mixtures thereof.Examples of natural cellulosics are cotton, linen, hemp, jute and ramie.Examples of manmade cellulosics are viscose, cellulose acetate,cellulose triacetate, rayon, cupro and lyocell. The above mentionedcellulosics can also be employed as blends of synthetic fibers such aspolyester, polyamide or acrylic fibers. The textile material may be yarnor knitted or woven or formed by any other means.

[0153] The cellulases of the invention, besides being especially usefulfor the treatment of fabric, are useful in general in any area requiringcellulase activity. In the pulp and paper industry, neutral cellulasescan be used, for example, in deinking of different recycled papers andpaperboards having neutral or alkaline pH, in improving the fiberquality, or increasing the drainage in paper manufacture. Other examplesinclude the removal of printing paste thickener and excess dye aftertextile printing, and as a treatment for animal feed. For example, ifthe intended application is improvement of the strength of themechanical pulp, then the 50K-cellulase, 20K-cellulase, 50K-cellulase Bor the protein-with-CBD preparations of the invention may provide one ormore of these proteins so as to enhance or facilitate the ability ofcellulose fibers to bind together. In a similar manner, in theapplication of pulp refining, the 50K-cellulase, 20K-cellulase,50K-cellulase B or protein-with-CBD preparations of the invention mayprovide one or more of these proteins at a level that enhance orfacilitate such swelling.

[0154] The invention is described in more detail in the followingexamples, These examples show only a few concrete applications of theinvention. It is self evident for one skilled in the art to createseveral similar applications. Hence the examples should not beinterpreted to narrow the scope of the invention only to clarify the useof the invention.

EXAMPLES Example 1 Shake Flask and Fermentor Cultivations

[0155] For maintenance, the strains ALKO4179, ALKO4124, ALKO4237,ALKO4265 and ALKO4125 were streaked on sporulation agar (ATCC medium 5,American Type Culture Collection, Catalogue of Filamentous Fungi, 18thedition, eds., S. C. Jong and M. J. Edwards, (1991): 1 liter contains 1g yeast extract, 1 g beef extract, 2 g tryptose, a trace amount ofFeSO₄, 10 g glucose and 15 g agar; the pH was 7.2. Agar slants wereincubated at 45° for 3-6 days.

[0156] For the applications tests of ALKO4237 (Examples 3 and 4), acolony was inoculated in 500 ml of the following mineral medium(Moloney, A. P. et al., Biotechnol. Bioeng. 25:1169 (1983)): 1 litercontains 15 g KH₂PO₄, 15 g (NH₄)₂SO₄, 2.4 ml of 1 M MgSO₄×7H₂O, 5.4 ml 1M CaCl₂, 20 g Solka floc, 15 g corn steep powder, 1 g yeast extract and10 ml 100× trace element solution 1, where 1 liter of 100× trace elementsolution 1 contains 0.5 g FeSO₄×7H₂O, 0.156 g MnSO₄×H₂O, 0.14 gZnSO₄×7H₂O and 0.49 g CoSO₄×7H₂O; the pH was adjusted to pH 6.5.Cultivation was performed at 45° C. for 3 days in a rotatory shaker (250rpm). Endoglucanase activity of about 20-25 nkat/ml was obtained.

[0157] Cellulase activity was routinely measured as endoglucanaseactivity according to Bailey, M. J. et al., Enzyme Microb. Technol.3:153 (1981)), using 1% (w/v) hydroxyethylcellulose, HEC (Fluka AG#54290) as a substrate. The assay conditions were, if not otherwisestated, pH 7.0 and 50° C. with a 10 minute reaction time. Oneendoglucanase unit (1 nkat=1 ECU) is defined as the amount of enzymethat produces reducing carbohydrates having a reducing powercorresponding to one nanomole of glucose in one second from HEC underthe assay conditions. However, with the purified enzymes described inExamples 9-12, the assay conditions of Bailey et al, Enzyme Microb.Technol. 3:153 (1981) exceed the linear range, and the assay wastherefore modified as described in Example 10. With every strain, thefilter paper activity assay (which measures the total hydrolysis ofcellulose and indicates the presence of cellobiohydrolase activity) waseither under the reliable detection limit or very low.

[0158] For the determination of pH and temperature dependency (Example2), as well as for the application tests of the strains ALKO4179,ALKO4124, ALKO4265 and ALKO4125 (Examples 3 and 4), colonies wereinoculated in 500 ml of the modified thermomedium B (G. Szakacs,Technical University of Budapest, Hungary): 1 liter contained 6 g Solkafloc, 6 g distiller's spent wheat grain, 3 g oat spelt xylan, 2 g CaCO₂,1.5 g soybean meal, 1.5 g (NH₄)₂HPO₄, 1 g barley bran, 0.5 g KH₂PO₄ 0.5g MgSO₄×7H₂O, 0.5 g NaCl, 0.5 ml trace element solution 1 (1 litercontains: 1.6 g MnSO₂, 3.45 g ZnSO₄×7H₂O, and 2.0 g CoCl₄×6H₂O) and 0.5ml trace element solution 2 (1 liter contains: 5.0 g FeSO₄×7H₂O and twodrops of concentrated H₂SO₄); the pH was adjusted to pH 6.5.Cultivations were performed at 45° C. for 3 days in a rotatory shaker(250 rpm). Because in thermomedium B the endoglucanase activities of thestrains ALKO4179, ALKO4124, and ALKO4237 were about 5 nkat/ml, culturefiltrates were concentrated about 10 fold in an Amicon concentratorusing a cut-off of 30 kDa. Endoglucanase activity obtained with ALKO4265 was about 20 nkat/ml and with ALKO 4125 30-40 nkat/ml.

[0159] The 1 liter fermentor cultivation of ALKO4179 was performed inthe following medium: 1 liter contained 10 g Solka floc, 3 g cellobiose,4 g corn steep powder, 1.5 g (NH₄)₂HPO₄, 0.3 g MgSO₄×7H₂, 0.5 g NaCl, 2g CaCO₃, 0.5 ml trace element solution 1 and 0.5 ml trace elementsolution 2, 0.5 g KNO₃, 0.3 g CaCl₂, 1 g Tween 80; the pH was adjustedto pH 6.5.

[0160] The 1 liter fermentor cultivation of ALKO4124 was performed inthe modified thermomedium B: 1 liter contained: 10 g Solka floc, 1 gRoth's xylan, 40 g whey, 30 g soybean meal, 2 g CaCO₃, 5 g (NH₄)₂SO₄,0.5 g KH₂PO₄, 1.0 g MgSO₄×7H₂O, 1.0 g NaCl, 1 g antifoam, 0.5 ml traceelement solution 1 and 0.5 ml trace element solution 2.

[0161] The 1 liter fermentor cultivation of ALKO4237 was performed inthe mineral medium mentioned above. 10% (v/v) inoculum was used. pH wasmaintained at pH 6.5±0.4 by the addition of ammonia [12.5% (v/v)] andphosphoric acid [17% (v/v)]. The fermentation temperature was 45° C. Thefermentor (Biostat M, B. Braun, Melsungen, Germany) was stirred at 400rpm and the air flow as 1 vvm. The endoglucanase activities obtainedwere the following: ALKO4179 about 40 nkat/ml, ALKO4124 about 90 nkat/mland ALKO4237 about 30 nkat/ml. ALKO4265 and ALKO4125 were not cultivatedin a fermentor.

[0162] ALKO4179, ALKO4124, ALKO4237 and ALKO4125 were cultivated in a100 liter pilot fermentor in media and conditions described above.Endoglucanase activities obtained were about 40 nkat/ml with ALKO4179and ALKO4237, about 90 nkat/ml with ALKO4124 and about 100 nkat/ml withALKO4125. Culture filtrates were concentrated 10-20 fold in a MilliporePUF 100 ultra filter and a Pellicon Us cassette concentrator using acut-off of 10 kDa.

Example 2 Determination of the pH and the Temperature Dependence of theEndoglucanase Activities in the Culture Filtrates

[0163] For the determination of pH and temperature dependence, thestrains ALKO4179, ALKO4124, ALKO4237, ALKO4265 and ALKO4125 were grownin the modified thermomedium B. Samples from the shake flaskcultivations (culture filtrates) were diluted in 50 mM McIlvain'sbuffers (50 mM citric acid-100 mM Na₂HPO₄) of pH range 4.5-8.5. Thefinal pH values of the culture filtrate buffer mixtures were 4.3, 5.4,6.3, 7.3, 8.1 and 8.7 for the strain ALKO4179; 4.3, 5.4, 6.4, 7.3, 8.1and 8.5 for the strain ALKO4124; 4.4, 5.3, 6.2, 7.1, 8.0 and 8.5 for thestrain ALKO4237; 4.3, 5.4, 6.3, 7.2, 8.1 and 8.5 for the strain ALKO4265and 4.3, 5.4, 6.4, 7.3, 8.1 and 8.5 for the strain ALKO4125. BSA wasadded as a protein carrier to the concentration of 100 μg/ml. PepstatinA and phenyl methyl sulphonyl fluoride (PMSF) were added as proteaseinhibitors at 10 μg/ml and 174 μg/ml, respectively. Endoglucanaseactivity was measured at each pH at 50° C. with 60 minutes reactiontime. The endoglucanase activity of ALKO4179 exhibited more than 90% ofits maximum in the pH range of about 4.5-7.5, the maximum activity wasdetected at about pH 5.4-6.3 (FIG. 1A). The endoglucanase activity ofALKO4124 exhibited more than 80% of its maximum activity in the pH rangeabout 5.5-7.5, the maximum activity was detected at about pH 6.4 (FIG.2A). The endoglucanase activity of ALKO4265 exhibited more than 80% ofits maximum activity in the pH range about 4.5-7.0, the maximum activitywas detected at about pH 5.5-6.5 (FIG. 4A). The endoglucanase activityof ALKO4237 exhibited more than 80% of its maximum in the pH range ofabout 4.5-6.0, the maximum activity was detected at about pH 5.3 (FIG.3A). The endoglucanase activity of ALKO4125 exhibited about 90% of itsmaximum in the pH range of about 4.5-7.5, the maximum activity wasdetected at about pH 6.5 (FIG. 5A).

[0164] For the temperature dependency determination of the endoglucanaseactivity, samples from the culture filtrates were diluted in 50 mMMcIlvain's buffer, pH 7.0. BSA was added as a protein carrier to theconcentration of 100 μg/ml. Pepstatin A and phenyl methyl sulphonylfluoride (PMSF) were added as protease inhibitors to 10 μg/ml and 174μg/ml, respectively. The final pH values of the culture filtrate buffermixtures were 7.3 (ALKO4179, ALKO4124 and ALKO4125) and 7.2 (ALKO4237and ALKO4265). Samples were incubated at 40° C., 50° C. and 60° C. for60 minutes. The maximum endoglucanase activity of ALKO4179 was detectedat 50° C. and 60° C., about 30% of the activity was retained at 40° C.(FIG. 1B). The maximum endoglucanase activity of ALKO4124 was detectedat 60° C., about 70% of the activity was retained at 50° C. and 30% at40° C. (FIG. 2B). The maximum endoglucanase activity of ALKO4237 wasdetected at 60° C., about 60% of the activity was retained at 50° C. and40% at 40° C. (FIG. 3B). The maximum endoglucanase activity of ALKO4265was detected at 60° C., about 50% of the activity was retained at 50° C.and 30% at 40° C. (FIG. 4B). The maximum endoglucanase activity ofALKO4125 was detected at 60° C., about 80% of the activity was retainedat 50° C. and 70% at 40° C. (FIG. 5B).

Example 3 Indigo Dye Release in Neutral Conditions

[0165] Cellulase preparations derived from the strains ALKO4179,ALKO4124, ALKO4237, ALKO4265 and ALKO4125 (Examples 1 and 2) were testedfor their ability to release dye in neutral conditions from the indigodyed cotton-containing denim fabric to give a stone-washed look.Commercial acid cellulase product Ecostone L (Primalco Ltd, Biotec,Finland) was used as a control.

[0166] Denim fabric was obtained from Lauffenmuehl (Germany). Testfabric was prewashed 10 min at 60° C. with Ecostone A 200 (1 ml/liter,Primalco Ltd, Biotec, Finland). The fabric was then cut into 12×12 cmswatches. The colour from both sides of the fabric swatches was measuredas reflectance values with the Minolta (Osaka, Japan) Chroma Meter CM1000R L*a*b* system.

[0167] Cellulase treatments were performed in LP-2 Launder-Ometer(Atlas, Ill., USA) as follows. About 7 g of denim swatches were loadedinto the 1.2 liter container that contained 200 ml of 0.05 Mcitrate/phosphate buffer at pH 7, or, 0.05 M citrate buffer at pH 5.2.0.06 ml of 10% Berol 08 (Berol Nobel AS, Sweden) was added as asurfactant.

[0168] A quantity of steel balls were added into each container to helpthe fiber removal. Finally the cellulase solutions were added to thecontainer as endoglucanase activity units (Example 1). The containerswere then closed and loaded into a 50° C. Launder-Ometer bath. TheLaunder-Ometer was run at 42 rpm for 2 hours.

[0169] After removing swatches from the containers they were soaked for10 min in 200 ml of 0.01 NaOH and rinsed for 10 min with cold water.Swatches were then dried for 1 hour at 105° C. and air dried overnight.The color from both sides of the swatches was measured with the MinoltaChroma Meter. Results from the color measurements of treated denimfabrics are shown in Table I. TABLE I Color Measurement of Denim FabricsTreated with Different Cellulase Preparations. Right side of the Reverseside of the Fabric Fabric Source of ECU/g delta delta Enzyme of fabric Lb E L b E pH 7* — — 2.3 0.8 3.1 1.5 0.1 0.9 ALKO4237 200 6.4 3.3 7.6 2.41.7 3.2 400 7.7 3.8 8.1 2.5 1.8 3.0 ALKO4179 200 5.5 2.4 6.4 2.8 1.9 3.0400 4.6 2.8 5.1 2.2 1.5 3.0 ALKO4124 200 4.8 2.8 6.1 3.3 1.2 2.5 400 NDND ND ND ND ND ALKO4125 200 4.0 2.7 5.6 2.3 1.5 2.3 400 ND ND ND ND NDND ALKO4265 200 2.2 3.6 5.1 −4.9 6.6 9.2 400 ND ND ND ND ND ND EcostoneL 200 1.6 0.7 1.6 0 1.7 1.6 400 1.6 0.9 1.8 −1.9 2.2 2.8 pH 5.2**Ecostone L 200 2.01 2.33 3.30 −2.74 4.35 4.71 400 3.19 2.76 4.35 −2.564.83 6.71

[0170] To compare the final look of the denim fabrics after washing withdifferent cellulase preparations, the color from both sides (reverseside and right side) of the fabrics was measured. From the results shownin Table I, it can be seen that the lightness and blueness units areclearly increased on the right side of the garments washed withpreparations of ALKO4179, ALKO4124, ALKO4237 and ALKO4125 cellulases,showing a good stone-washed effect. The blueness unit was also increasedon the right side of the fabric washed with the ALKO4265 preparation butthere was no increase in the lightness unit. This is probably becausethe enzyme does work at this pH but at the same time causes a lot ofbackstaining. There was no stone washing effect on the fabric withcommercial acid product Ecostone L at pH 7 at this ECU activity.

[0171] In this study, backstaining on the reverse side of the fabric isused as an indication of the degree of backstaining on the right side ofthe fabric. To quantify the level of backstaining, the color wasmeasured on the reverse side of the fabric before and after thecellulase treatment. As shown in Table I, when the ECU amounts are thesame, there was practically no backstaining in the fabrics treated withthe ALKO4179, ALKO4124, ALKO4237 and ALKO4125 preparations when comparedto the fabrics treated with ALKO4265 or Ecostone L (pH 5.2 and 7)preparations.

Example 4 Dye Release in Neutral Conditions, No Berol

[0172] The experimental set-up was as described in Example 3 except thatno Berol was used. Results from the color measurements of treated denimfabrics are shown in Table II. TABLE II Color Measurement of DenimFabrics Treated with Different Cellulase Preparations - no Berol. Rightside of the Reverse side of the Source Fabric Fabric of ECU/g deltadelta Enzyme of fabric L b E L b E pH 7* — — 2.1 0.5 2.2 1.7 −1.1 2.0ALKO4237 200 5.5 3.1 7.0 1.8 2.3 3.5 ALKO4179 200 4.4 3.2 5.6 1.4 2.22.7 ALKO4124 200 4.2 2.9 5.0 1.1 2.0 2.4 ALKO4125 200 3.5 2.6 4.4 1.61.4 2.5 ALKO4265 200 3.3 3.3 5.3 −5.7 6.6 10.0 Ecostone L 200 1.4 0.91.7 0.3 1.4 1.8 400 1.4 0.8 1.7 −0.1 1.7 1.8 pH 5.2** Ecostone L 200 2.02.1 2.9 −4.0 4.8 5.4

[0173] When compared with results obtained with the inclusion of Berol(Example 3), the data in Table II show that almost the samestone-washing effect can be achieved with the ALKO4179, ALKO4124,ALKO4237 and ALKO4125 cellulase preparations in the absence of thehelping agent Berol.

Example 5 Backstaining in Denim Wash with Different Cellulases

[0174] In the literature, it is reported that backstaining is dependenton pH and/or the type of enzyme. However, as shown herein, it was foundthat backstaining depends only indirectly on pH (FIGS. 6A and 6B and 7Aand 7B).

[0175] Two neutral cellulase preparations from ALKO4237 and fromALKO4265 and acid cellulase product Ecostone L were studied in smallscale denim wash with an equal enzyme dosage at pH 5 and pH 7. Thestonewash effect was determined by measuring the increase of lightnessand blueness as reflectance units on the right side of the fabric andbackstaining (redeposition of indigo on the surface of fibers) wasdetermined as blueness increase and lightness decrease on the reverseside. At pH 7, the neutral cellulases from ALKO4237 caused a clearincrease in lightness and blueness on the right side and no backstainingwas observed (FIGS. 6A and 6B). A similar stonewash effect was found atpH 5 but with a slight backstaining. At pH 7, the other neutralcellulase, ALKO4265, brightened blueness on the right side butbackstained intensively on the reverse side. At pH 5 similar effectswere obtained with both ALKO4265 and ALKO4237 preparations. At pH 7, theacid cellulase did not backstain or impart a lightness on the right side(when using similar endoglucanase activity dosages as with ALKO4265 andALKO4237, FIGS. 7A and 7B, 1× dosage), probably because it did not workat this pH. On the other hand, at pH 5, lightness and blueness wereincreased on the right side and backstaining was clearly perceptible onthe reverse side. Based on these results, backstaining can occur at bothpH values depending on the cellulase preparation used.

Example 6 Use of the Neutral Cellulase-Containing Enzyme Preparations inBiofinishing of Cotton-Containing Woven Fabric

[0176] 100% cotton woven fabric was subjected to treatment with ALKO4237(Example 1) and ALKO4467 cellulases in Launder-Ometer. ALKO4467 is aUV-mutant with higher cellulase activity derived from ALKO4125.

[0177] 100% cotton woven fabric (obtained from Pirkanmaan Uusi VärjäämöLtd) was pretreated as in Example 7. The cellulase treatment conditionswere as described in Example 3 except that no Berol was used and theliquid ratio was 1:15 (volume of liquid per weight of fabric).Cellulases were dosed as ECU activity units (Example 1).

[0178] The following methods were used for evaluation of the effect ofthe enzyme preparations in biofinishing of cotton fabric: Weight loss ofthe treated fabrics was defined as percentage from weight of the fabricbefore and after the test (before weighing the fabrics were conditioningin a atmosphere of 21+2° C. and 50+5% RH). Evaluation of the surfacecleaning effect of the enzyme treated fabrics was performed by a panelconsisting of three persons. The fabrics were ranked on a score from 1to 5, where 5 gave a clean surface. The Martindale Rubbing method(SFS-4328) was used for evaluation of pilling. Pilling was evaluated bya panel after 200 and 2000 cycles of abrasion (1=many pills, 5=nopills).

[0179] In Table III is shown that treatment of the cotton fabric withALKO4237 and ALKO4467 cellulase preparations results in a good surfacecleaning and marked reduction in the pilling tendency at both pH 5 and7. TABLE III Weight loss, surface cleaning effect and pilling tendencyof the cotton fabrics treated with neutral cellulases in Launder-Ometer.weight surface pilling pre- dosage time loss cleaning 200 2000 parationECU/g h pH % effect cycles cycles — — 1 5 0 1.0 1.0 1.0 ALKO4237 200 1 52.3 3.5 4.0 3.8 ALKO4237 400 1 5 3.2 3.5 4.0 3.8 ALKO4467 200 1 5 1.22.5 3.7 3.4 ALKO4467 400 1 5 1.9 2.8 3.7 3.4 — — 2 5 0.1 1.0 1.0 1.0ALKO4237 200 2 5 4.4 4.0 4.2 4.1 ALKO4237 400 2 5 6.0 4.3 4.2 4.3ALKO4467 200 2 5 3.0 3.5 4.0 3.8 ALKO4467 400 2 5 4.0 3.8 4.0 3.9 — — 17 0 1.0 1.0 1.0 ALKO4237 200 1 7 2.5 3.0 3.7 3.5 ALKO4237 400 1 7 3.84.0 4.0 3.9 ALKO4467 200 1 7 0.8 2.0 3.5 3.3 ALKO4467 400 1 7 1.4 2.03.6 3.7 — — 2 7 0.1 1.0 1.2 1.1 ALKO4237 200 2 7 4.8 4.0 3.8 4.0ALKO4237 400 2 7 6.0 4.3 4.0 4.3 ALKO4467 200 2 7 2.2 2.5 4.0 3.4ALKO4467 400 2 7 3.0 3.3 3.8 3.7

Example 7 Use of the Neutral Cellulase-Containing Enzyme Preparations ofthe Invention in Biofinishing

[0180] 7a. Use of Enzyme Preparations in the Biofinishing of WovenFabric and Knit.

[0181] 100% cotton woven fabric or 100% cotton knit are subjected totreatment with the cellulases of the invention (Example 1) in asemi-industrial drum washer (Esteri 20 HS-P). The treatment conditionsare as follows:

[0182] A. Pretreatment (Only for Woven Fabrics)

[0183] 60° C., 10 minutes, Ecostone A200 (Primalco Ltd, Biotec, Finland)1 ml/l water.

[0184] B. Enzyme Treatment

[0185] temperature 50-60° C., pH 7;

[0186] liquid ratio 5-20:1 (volume of liquid per weight of fabric);

[0187] treatment time 20-90 minutes, preferably 30-60 minutes; and

[0188] enzyme dosage 50-900 nkat/g fabric or knit, preferably 200-600nkat/g fabric or knit.

[0189] C. “After-Washing” Treatment

[0190] 40° C., 10 minutes, alkaline detergent

[0191] D. Drying Treatment

[0192] The following standard methods are used for evaluation of thesurface cleaning effect of enzyme preparations: The Martindale RubbingMethod (SFS-4328) and the Laundering Durability Test (SFS-3378).Treatment with the cellulase preparations of the invention results in asurface cleaning effect, an improvement in the softness and smoothnessof the fabric and knit and a reduction in the pilling tendency.

[0193] 7b. Use of Enzyme Preparations in the Finishing of LyocellFabrics and Knits.

[0194] The cellulase preparations of the invention can be used infibrillation control and different finishing processes of 100% lyocellfabrics and knits and blends thereof. The following treatment conditionsin semi-industrial drum washer (Esteri 20 HS-P) are used in order tocreate the peach effect on lyocell fabric:

[0195] A. Sodium carbonate 2.5 g/l; 60° C., treatment time of 60minutes;

[0196] B. Rinse;

[0197] C. Enzyme treatment: temperature of 50-60° C., pH 7, liquid ratio5-20:1, treatment time 40-120 minutes, preferably 45-90 minutes, and anenzyme dosage of 100-1500 nkat/g fabric, preferably 400-800 nkat/gfabric;

[0198] D. Aftertreatment: Alkaline detergent wash at 40° C. for 10minutes;

[0199] E. Rinse; and

[0200] F. Dry.

[0201] The result is a peach skin effect.

Example 8 Use of Enzyme Preparations in Biostoning

[0202] Denim garments were subjected to treatment with the neutralcellulase preparations (Example 1) in a semi-industrial drum washer(Esteri 20 HS-P) to give the garments a stonewashed appearance. About1.0 kg of denim garments (contained two different kinds of fabric) wereused per machine load.

[0203] The treatment conditions were as follows.

[0204] A. Desizing. 100 liters water, 60° C., 10 minutes; 100 mlEcostone A200 (Primalco Ltd, Biotec, Finland).

[0205] B. Cellulase Treatment 100 liter water, 50° C., 45 minutes; 10 gBerol 08 (Berol Nobel AS, Sweden); 30 g citric acid+128 g Na₂HPO₄×2H₂Oto give pH 7.

[0206] Neutral cellulase preparations were dosed as endoglucanaseactivity units (ECU, Example 1):

[0207] 1. ALKO4237, 260 ECU/g of garment

[0208] 2. ALKO4179, 260 ECU/g of garment

[0209] 3. ALKO4124, 300 ECU/g of garment

[0210] 4. ALKO4125, 250 ECU/g of garment

[0211] C Afterwashing. Alkaline detergent wash, 40° C., 10 minutes.

[0212] D. Drying.

[0213] The results were evaluated by visual appearance of the garmentsand by measuring the color as reflectance values with the Minolta ChromaMeter CM 1000R L*a*b system (Table IV). A good stonewashed effect wasobtained with all these cellulase-treated garments. No backstaining(examined on the inside of the garment) could be seen visually in any ofthese cellulase-treated garments.

[0214] From the results of the color measurements shown in Table IV, itcan be seen that the lightness and blueness units are clearly increasedon the outside of the garments washed with the neutral cellulasepreparations, showing a good stonewashed effect. TABLE IV ColorMeasurement of Denim Garments with Different Cellulase PreparationsSource of Outside of the garment Inside of the garment Enzyme L b L b A.Fabric 1 untreated 24.1  −8.5 57.1    0.17  washed without 21.4 −14.054.5 −4.3 cellulase ALKO4237 26.7 −17.3 56.5 −4.9 ALKO4179 26.8 −17.056.3 −4.5 ALKO4125 28.0 −17.4 57.8 −4.1 ALKO4124 26.4 −17.5 57.1 −4.8 B.Fabric 2 untreated 22.5  −8.3 57.6    0.66  ALKO4237 25.0 −16.3 56.1−4.3 ALKO4179 25.0 −15.8 55.4 −4.4 ALKO4125 26.7 −17.0 56.8 −4.0ALKO4124 25.6 −17.0 56.4 −4.0

Example 9 Purification of Neutral Cellulases

[0215] Concentrated growth medium from ALKO4237 was fractionated at 7°C. on DEAE Sepharose CL6B with a linear gradient from zero to 0.5 M NaClin 25 mM Tris/HCl pH 7.2. Four peaks of endoglucanase activity at pH 4.8were found. Peak I, containing about 10% of the recovered ECU, eluted atabout 150 mM NaCl, Peak II (about 30% of ECU) at 230 nM NaCl, Peak III(about 20% of ECU) at 270 mM NaCl and Peak IV (about 40% of ECU) at 320mM NaCl. Table V shows the results when these peaks were tested fortheir utility in biostoning at neutral pH and 50° C.

[0216] These results show that on both an ECU basis and a total proteinbasis, Peak II was more effective than any other peak or than theunfractionated concentrate. A mixture of Peaks I and II containing 70ECU of each/g denim was also tested. This resulted in an L (right) valueof 7.3 and b (reverse) of 2.5. Thus, this mixture was more effectivethan either peak alone.

[0217] The purification procedure was scaled up to obtain homogenoussamples of some of the desired proteins in these peaks. ConcentratedALKO4237 growth medium (4.5 liters) was fractionated with ammoniumsulphate. The proteins that precipitated between 17 g and 42 g ofammonium sulphate per 100 ml of concentrate were suspended in 0.9 literof 25 mM Tris/HCl pH 7.2 containing 0.25 mM EDTA and then diluted withwater to a conductivity of 4 mS/cm and adjusted with 1 M NaOH to pH 8.0.The resulting solution (about 45 liters) was pumped at 150 m/min througha 6.3 liter column of DEAE-Sepharose FF™ at room temperature. Peak Iendoglucanase activity did not bind under these conditions. Boundproteins were eluted at 110 ml/min with a linear gradient from 0.0 to0.5 M NaCl in 20 liters of 25 mM Tris/HCl pH 7.7 containing 0.25 mMEDTA. Peak II endoglucanase eluted at about 14 mS/cm. Instead of theseparate Peaks III and IV seen with small scale separations in DEAE inthe cold room, a single peak, called Peak III/IV, eluted at about 25mS/cm. TABLE V Indigo Dye Release by DEAE-Sepharose ™ Pools in NeutralConditions ADDITION None Concentrate Peak I Peak II Peak III Peak IVECU/g 0 100 200 310 185 340 97 260 95 190 mg/g 0 10 20 41 9 26 24 46 510 L (right) 2.9 5.2 7.0 5.5 7.3 10.3 4.4 5.8 3.9 4.3 b (reverse) 0.42.6 3.5 3.5 2.9 2.5 0.9 1.4 0.1 0.5 # reverse side (i.e., backstaining).The fabric was washed in the LP-2 Launder-Ometer and then measured withthe Minolta ChromaMeter, as # described in Example 3, except that noBerol was used and the buffer that was used was 0.05 M Mellvaine pH 7(see Data for Biochemical # Research, Dawson, R., et al., eds., 1969,Oxford Univ. Press). The dosage is shown as both ECU/g of denim and mgprotein/g of denim.

[0218] Proteins in Peak II (3.5 liters) were precipitated with ammoniumsulphate (450 g/liter) and suspended in 170 ml 25 mM PIPES/KOH pH 6.0containing 1 mM EDTA. Portions of this material were transferred to 25mM sodium acetate pH 4.0 containing 1 mM EDTA by gel-filtration on a5×29 cm column of G25 Sephadex™ (coarse) and then fractionated onSP-Sepharose™. FIG. 8 shows the result that was obtained when 11.7 g ofthese proteins was applied to a 4.5×31 cm column of SP-Sepharose™ in 25mM sodium acetate pH 4.0 containing 1 mM EDTA at 150 ml/h and the columndeveloped at 75 ml/h with a linear gradient from 0.0 to 0.4 M NaCl in3.4 liters of the same buffer. Most of the endoglucanase eluted at 0.2 MNaCl. The modified assay described in Example 10 was used. When activefractions were stored at 7° C., a crystalline precipitate appeared inthem and contained nearly all the endoglucanase activity. Activefractions (15 ml) in which crystallization was slow, were induced toform crystals by seeding with 30 μl of suspension from fractions alreadycontaining crystals. After 2 to 3 days, the crystals were collected bycentrifugation, washed with 25 mM PIPES/KOH pH 6.0 containing 1 mM EDTAand disolved in 25 mM Tris/HCl pH 7.2 containing 0.25 mM EDTA. Analysisby SDS-PAGE showed the washed crystals contained a virtually homogenousprotein with an apparent molecular mass close to 20 kDa (the error inSDS-PAGE estimations of molecular mass is at least ±10%, and may be muchgreater for unusual proteins). This protein is called the 20K-cellulase.Contaminating protein could also be removed by gel-filtration on G50Sephadex™ in 50 mM PIPES/KOH pH 6.0 containing 1 mM EDTA. An example ofthis is shown in FIG. 9, where unwashed crystals were purified bygel-filtration. The endoglucanase activity co-eluted with the 20 kDaprotein well after the cytochrome c (11.2 kDa) volume, showing that this20 kDa protein is abnormally retarded by interaction with Sephadex™.

[0219] Proteins in Peak III/IV were precipitated with ammonium sulphateand transferred to 25 mM sodium acetate pH 4.0 containing 1 mM EDTA inthe same way as described for the Peak II proteins. Upon transfer to 25mM sodium acetate pH 4.0, a large precipitate formed and was discarded.The active supernatant was fractionated on SP-Sepharose™. At low proteinloading (e.g. 200 mg protein to a 2.5×11 cm column as shown in FIG. 10,most of the endoglucanase activity bound to the column and was elutedwith a NaCl gradient at about 50 mM NaCl. This active peak was followedby a second peak of inactive protein.

[0220] SDS-PAGE analysis showed that the active and inactive peaks bothcontained several proteins, including proteins with apparent molecularmasses close to 50 kDa that could not be distinguished from each otherby SDS-PAGE. Both peaks were further purified by chromatography onPhenyl Sepharose™.

[0221] The active fractions (fractions 15 to 18 in FIG. 10) were pooled,adjusted to 50 mM PIPES/KOH pH 6.0 (by addition of 0.25 M PIPES/KOH pH6.0) and 15 g % ammonium sulphate (by addition of solid ammoniumsulphate) and applied to a 1.5×8.5 cm column of Phenyl Sepharose™equilibrated with 25 mM PIPES/KOH pH 6.0 containing 1 mM EDTA and 15 g %of ammonium sulphate. The column was developed with a linear gradientfrom 15 to 0 g % ammonium sulphate in 104 ml of 25 mM PIPES/KOH pH 6.0.After the end of the gradient, the column was further washed with 25 mMPIPES/KOH pH 6.0. Two protein peaks eluted on the gradient, first asmall peak of inactive protein and then a major peak containing most ofthe endoglucanase activity. SDS-PAGE analysis (FIGS. 11A and B) showedthat both peaks contained essentially homogenous proteins with apparentmolecular masses close to 50 kDa (i.e., they migrate slightly slowerthan the BioRad prestained ovalbumin standard, which has an apparentmolecular mass of 47 kDa). These two proteins could not be distinguishedby the inventors' SDS-PAGE analyses, even when they were run together asmixtures. The protein in the active peak was called 50K-cellulase andthe protein in the inactive peak was called 50K-protein B. Largeramounts of 50K-cellulase B were obtained by fractionation of the second(and inactive) peak eluted from SP-Sepharose™ (fractions 19 to 23 inFIG. 10) on Phenyl Sepharose™ in exactly the same way as described abovefor the active fractions.

[0222] Production of still larger amounts of 50K-cellulase and50K-cellulase B was facilitated by overloading the SP-Sepharose™ column.For example, when 15 g of protein was applied to a 4.5×31 cm column ofSP-Sepharose™, instead of binding to the column, the 50K-cellulase wasapparently displaced by more strongly bound proteins, and eluted beforethe NaCl gradient. This material was already highly purified, andhomogenous 50K-cellulase was isolated from it by chromatography onPhenyl Sepharose™ as described above.

[0223] In order to speed up the purification of larger amounts of50K-cellulase the SP-Sepharose and Phenyl Sepharose columns werereversed. After adjusting the ammonium sulphate concentration to about15 g %, the proteins precipitated in Peak III/IV were applied intoPhenyl Sepharose as described before. With high overloading (e.g. 17 gof protein applied to a 3.2×25 cm column of Phenyl Sepharose) most ofthe total protein ran through the column, but 50K-cellulase (containingmost of the endoglucanase activity) was bound and eluted at the end oflinear gradient from 15 to 0 g % of ammonium sulphate in 25 mM PIPES/KOHpH 6.0. Western analysis with a rabbit antiserum recognizing50K-cellulase B showed that the 50K-cellulase B eluted just before50K-cellulase. Further purification was achieved by fractionation onSP-Sepharose as described earlier. In this reversed order ofSP-Sepharose and Phenyl Sepharose the proteins in Peak III/IVprecipitated with ammonium sulphate could be applied directly to thenext purification step without removing salt. The large proteinprecipitate, which appeared upon transfer of the concentrated proteinsin Peak III/IV directly into 25 mM sodium acetate pH 4.0 forSP-Sepharose, could also be avoided this way. As the 50K-cellulase onlyjust binds to SP-Sepharose, the preceeding fractionation on PhenylSepharose markedly reduced the apparently interfering total protein loadon SP-Sepharose.

[0224] 50K-cellulase and 50K-cellulase B were each tested in theLaunder-Ometer to see if they are responsible for the beneficial effectsof Peak IV reported in Example 10. Both proteins were found to havebeneficial effects (Table VI). At the low concentrations used in thisexperiment, they did not themselves increase the release of indigo dyefrom the outer face of the denim (i.e., L_(right) did not increase) butthey effectively decreased the back-staining of dye onto the inner faceof the denim (L_(reverse) became more positive and b_(reverse) becamesmaller) especially when used together with 20K-cellulase.

[0225] The 20K-cellulase performed well in Launder-Ometer tests at pH 5as well as at pH 7. At pH 5, 0.2 mg of 20K-cellulase per g of denimincreased L_(right) from 3.2 to 5.2. Addition of 50K-cellulase at 0.1 mgper gram of denim together with the 20K-cellulase also decreased thebackstaining at pH 5 (L_(reverse) and b_(reverse) changed from 0.0 and2.6 with 20K-cellulase alone to 1.3 and 1.5, respectively, with themixture of 20K- and 50K-cellulases). TABLE VI Indigo Dye Release by20K-cellulase, 50K-ceIlulase and 50K-cellulase B Dose Sample (mg/g)L_(right) L_(reverse) b_(reverse) Buffer alone — 2.8 −0.6   1.620K-cellulase 0.18 5.6 −1.0   4.0 0.09 4.8 −1.5   3.3 50K-cellulase 0.152.6 −0.3   1.0 0.075 3.0 0.4 1.3 50K-cellulase B 0.31 2.8 1.3 0.8 0.152.7 1.5 0.5 20K-cellulase + 0.18 + 0.075 5.6 0.3 2.5 50K-cellulase0.09 + 0.075 5.1 0.3 2.1 20K-cellulase + 0.18 + 0.15  4.7 0.0 3.050K-cellulase B

Example 10 Properties of the 20K-Cellulase

[0226] Although polyclonal antibodies prepared against cellulasespurified from Trichoderma reesei (designated anti-EGI, anti-CBHI andanti-CBHII antibodies) recognized proteins in the ALKO4237 growthmedium, there was only a very weak cross-reaction with pure20K-cellulase under the same conditions of Western blot analysis.

[0227] When growth medium from ALKO4237 was probed on Western analysiswith antiserum raised in rabbits against pure 20K cellulase, a strongband at about 35 kDa was observed in addition to the 20 kDa band. Noapparent endoglucanase activity could be detected for this 35 kDaprotein. Also, a weaker band was seen immediately ahead of the 20 kDaband (FIG. 14).

[0228] ALKO4124 gave an almost identical pattern as ALKO4237, indicatingthat this and other fungi probably contain cellulases very similar tothe 20K-cellulase of the present invention.

[0229] Amino acid sequences of tryptic peptides derived from20K-cellulases are shown in FIG. 17.

[0230] Purified 20K-cellulase performed well in biostoning at neutral pHwithout the addition of other enzyme activities as shown in Table VII.TABLE VII Biostoning by Purified 20K-cellulase Addition Dosage L_(right)b_(right) L_(reverse) b_(reverse) Buffer 0.0 3.6 0.1 0.5 0.6 20K 0.728.9 2.9 −1.1 4.7 20K 0.25 6.0 2.3 −0.5 3.6 20K 0.07 5.3 1.7 −0.4 2.9Whole medium 20 6.1 2.8 −2.9 5.5 # the unfractionated ALKO4237concentrated growth medium.

[0231] Compared to the unfractionated medium, 20K-cellulase resulted inthe same degree of lightening (L_(right)=6.0-6.1) at {fraction (1/80)}ththe protein dosage. Further, there was less backstaining onto thereverse side face of the fabric (L_(reverse)−0.5 compared to −2.9 andb_(reverse)=3.6 compared to 5.5). Fabric treated with 20K-cellulase hadan agreeable soft texture.

[0232] Although 20K-cellulase performed surprisingly well without otheradditions, even better fabric appearance and texture resulted when 20Kwas used together with the DEAE-Sepharose pools I, III or IV (TableVIII). TABLE VIII Synergy in Biostoning Between 20K-cellulase andEndoglucanase Pools Eluted from DEAE-Sepharose Addition Dosage L_(right)b_(right) L_(reverse) b_(reverse) Buffer 0.0 3.8 0.2 −0.7 1.5 20K 0.185.8 2.3 −2.2 5.5 Pool I 15 5.1 1.9 −3.1 5.7 Pool III 47 5.2 1.6 −0.1 2.6Pool IV 14 5.6 0.9 0.4 1.8 20K + Pool I 15.18 7.1 2.8 0.7 3.3 20K + PoolIII 47.18 7.6 3.1 −1.7 5.3 20K + Pool IV 14.18 8.6 2.6 0.8 3.2 Wholemedium 20 5.7 2.4 −4.1 5.9

[0233] The mixtures of 20K-cellulase with Pools I, III and IV causedmore lightening (increased L_(right)) than either component alone. Atleast for the combination of 20K-cellulase with Pool IV, it is clearthat this is because of synergy and not merely an additive effect.Further, the backstaining with all mixtures was actually less(L_(reverse) more positive, b_(reverse) less) than the backstainingobserved with 20K-cellulase alone. The combination of 20K with Pool IVwas particularly effective. Pool IV contains many proteins, one of which(a 50 kDa polypeptide) copurifies with endoglucanase activity duringchromatography of Pool IV on Sephadex G100 and S-Sepharose. While goodbiostoning is achieved with 20K-cellulase alone, better results arepossible with 20K-cellulase plus one or more proteins purified from PoolIV. Biostoning with mixtures of the 20K-cellulase and the 50K-cellulaseand the 50K-cellulase B purified from Pool III/IV have already beenpresented (Table VI in Example 9). Therefore, the present invention isnot limited to the use of only the 20K-cellulase. Other proteins in theALKO4237 medium are useful alone or in suitable combinations.

[0234] In the standard endoglucanase assay described by Bailey et al.(1981, loc. cit.), the enzyme amount is chosen that produces, in 10 minand pH 4.8 (0.05 M Na-citrate buffer), about 0.6 mM reducing equivalentsfrom 1% hydroxyethylcellulose, resulting in a final absorbance change(ΔA₅₄₀) of between 0.2 and 0.25. This far exceeds the range in whichΔA₅₄₀ is proportional to the amount of 20K-cellulase.

[0235] Therefore, the procedure was modified as follows. Enough enzymewas used to produce about 0.2 mM reducing equivalents in 10 min in 0.05M HEPES buffer (pH 7.0). To reach the threshold concentration ofreducing equivalents above which color is formed in the DNS system, 0.12mM glucose was freshly added to the stock DNS reagent. This method(called the “modified” method) was used when characterizing theendoglucanase activity of the 20K-cellulase and also the 50K-cellulase.With 1% hydroxyethylcellulose as substrate, the range in which ΔA₅₄₀ isproportional to the amount of 20- and 50K-cellulase is relativelynarrow, and so 2% carboxymethylcellulose was taken as an altenativesubstrate. With 2% carboxymethylcellulose, the range of linearcorrelation between ΔA₅₄₀ and the amount of 20K- and 50K-cellulase wasbroader than with 1% hydroxyethylcellulose. The endoglucanase activitydetermined with 2% carboxymethylcellulose was about 8-10-fold for20K-cellulase and about 50-fold for 50K-cellulase compared with thatdetermined with 1% hydroxyethylcellulose.

[0236] No activity of 20K-cellulase was detectable for4-methylumbelliferyl-β-D-lactoside, a characteristic substrate ofcellobiohydrolases. The activity towards filter paper was also very low,but detectable.

[0237] The 20K-cellulase was relatively heat stable. It was incubated at7 μg/ml and 100° C. in 25 mM Tris-HCl, 0.2 mM EDTA, for 30 or 60 min.and then assayed at pH 7.0 and 50° C. 52% and 35% respectively, of theendoglucanase activity remained at pH 7.2.40% and 22%, respectively,remained at pH 8.8. (These pH values were measured at room temperature;the actual pH at 100° C. is somewhat lower.) At 80° C., pH 7.2, 70% ofthe activity remained for 60 min.

[0238] These results indicate that the enzyme is suitable forapplications in which it may be (e.g., accidentally) exposed to elevatedtemperatures. As well as being resistant to irreversible inactivation athigh temperatures, the enzyme exhibited an optimum temperature of 70° C.during 10 min. assays at pH 7.0 (FIG. 15). The decreased activityobserved above 70° C. was mainly due to a reversible change in enzymeconformation: the enzyme recovered most of its activity when returned to50° C.

[0239] At 0.50° C., the 20K-cellulase exhibited 80% or more of itsmaximum activity throughout the pH range 4 to 9, and nearly 50% at pH10. This was the case in both 10 min. (FIG. 16A) and 60 min. (FIG. 16B)assays. These figures also show the pH dependence of the enzyme at 70°C. With 10 min. assays, the enzyme was more active at 70° C. than it wasat 50° C. over the range pH 4.5 to 8 and about equally active at pH 10(FIG. 16A). With 60 min. assays (i.e., approaching commercialconditions), the enzyme was more active at 70° C. than it was at 50° C.between pH 5.5 and 7.5. However, it was only slightly less active at 70°C. than at 50° C. up to pH 10. In practice, this means that the enzymecan be used equally well over a wide range of pH and at temperatures upto at least 70° C.

Example 11 Properties of the 50K-Cellulase

[0240] Pure 50K-cellulase had both endoglucanase activity (againsthydroxyethylcellulose) and cellobiohydrolase activity (against4-methylumbelliferyl-β-D-lactoside, assayed essentially as described byvan Tilbeurgh et al, in Methods in Enzymology [1988] vol. 160, pp45-59). A sample of the pure enzyme with an A₂₈₀ of 1.8 contained 2030ECU/ml and 300 PCU/ml at pH 7.0 and 50° C. (one PCU is the amount ofactivity that liberates 1 nmol of methylumbelliferone per second).

[0241] In Western analyses, 50K-cellulase was strongly recognized byantiserum (KH 1057) raised against endoglucanase I of T. reesei, butonly weakly by antisera (KH 1050 and KH 1053, respectively) againstcellobiohydrolases I and II of T. reesei. It was not recognized by theantiserum raised against 20K-cellulase (FIG. 14). When the growth mediumof ALKO 4237 was probed in Western analyses with rabbit antiserum raisedagainst 50K-cellulase itself, only one obvious band (which had amolecular mass between 33 and 47 kDa) was seen in addition to the verystrong band at about 50 kDa.

[0242] The apparent molecular mass of 50K-cellulase by SDS-PAGEdecreased by about 2 to 5 kDa when the protein was treated withendoglycosidase H_(f), indicating that the enzyme contains carbohydrateremovable by this endoglycosidase.

[0243] 50K-cellulase was unusually resistant to tryptic digestion,indicating that it has an unusually stable structure. However, it wascleaved by treatment with cyanogenbromide, and the resulting fragmentscould then be digested with trypsin or with lysylendopeptidase C.Sequences of some of the peptides so obtained are shown in Table IX.TABLE IX Sequences of peptides isolated from the 50K- cellulase(uncertain residues in lower case) #507 VYLLDETEHR #509 XXLNPGGAYYGT#563 MsEGAECEYDGVCDKDG #565 NPYRVXITDYYGNS #603 DPTGARSELNPGGAYYGTGYXDAQ#605 XXVPDYhQHGVda #610 NEMDIXEANSRA #611 LPXGMNSALYLSEMDPTGARSELNP #612VEPSPEVTYSNLRXGEIXgXF #619 DGCGWNPYRVvITtDYYnN #620 LPCGMXSALY #621ADGCQPRTNYIVLDdLlHPXXQ

[0244] The 50K-cellulase is a stable enzyme that exhibits endoglucanaseactivity over a wide range of pH values and at high temperatures, so itis suitable for use in many industrial conditions. At pH 7.0 and with 60min reaction times, it has an optimum temperature between 65 and 70° C.,and even with this long reaction time still exhibits, at 75° C., 50% ofthe activity observed at 50° C. (FIG. 12).

[0245] With 60 min reaction times, the pH optimum was very broad at 50°C., with essentially constant activity between pH 4.4 and 7.0, andactivities at pH 9 and 10 equal to 50% and 30%, respectively, of that atpH 7.0. At 70° C., there was a clear optimum at pH 6, and, between pH 5and 7, the activity (with 60 min reaction times) was 3-fold or moregreater than that at 50° C. However, at pH 4.4 and pH values above 8,the activity was greater at 50° C. than at 70° C. (in 60 min assays),suggesting that the stability of the enzyme decreases at 70° C. rightside the pH range 5 to 7.5. The pH-dependence is illustrated in FIG. 13.

Example 12 Properties of 50K-Cellulase B

[0246] No detectable endoglucanase activity could be measured for the50K-cellulase B (previously called 50K-protein B) withhydroxyethylcellulose or carboxymethylcellulose. At acidic pH, the50K-cellulase B had a low cellobiohydrolase activity, which (measuredwith 4-methylumbelliferyl-β-D-lactoside) at pH 5 was less than 0.1% ofthat of the 50K cellulase. In addition, the 50K-cellulase B had adetectable activity towards filter paper at pH 4.8 and acid swollen,amorphic Solca Floc-cellulose at pH 5 and 7 used in enzyme activitydeterminations.

[0247] In Western analyses, 50K-cellulase B was strongly recognized byantiserum (KH1050) raised against cellobiohydrolase I of T reesei, butonly weakly by antisera against cellobiohydrolase II or endoglucanase Iof T. reesei or against the 50K-cellulase. It was not recognized byantiserum raised against the 20K-cellulase (FIG. 14). Table X showssequences of peptides isolated from 50K-cellulase B. TABLE X Sequencesof peptides isolated from the 50K- cellulase B (uncertain residues inlower case) #534 vGNPDFYGK #535 FGPIGSTY #631 LSQYFIQDGeRK #632FTVVSRFEENK #636 HEYGTNVGSRFYLMNGPDK

Example 13

[0248] Stability of Neutral Cellulases in Different Detergents

[0249] Stability of the neutral cellulase preparations were tested inthree different detergent solutions. The detergent solutions were OMO®Total (or OMO® Neste, Lever UK), OMO® Color (Lever S.A.) and ColourDetergent Liquid (Unilever, The Netherlands). The tested cellulasepreparations were ALKO4125, ALKO4179, ALKO4237 and ALKO4265 (Example 1)concentrated culture filtrates and purified 20K- and 50K-cellulases fromthe ALKO4237 strain (Example 9).

[0250] Cellulase preparations were incubated at 40° C. in 0.25%detergent solutions. The activity against hydroxyethylcellulose (ECU/ml,Example 1) was measured (pH 7, 50° C.) from samples taken after 5-30minutes incubation.

[0251] The tested preparations were as follows:

[0252] Culture filtrates:

[0253] ALKO4125: 780 ECU/ml (pH 7, 50° C.)

[0254] ALKO4179: 830 ECU/ml

[0255] ALKO4265: 760 ECU/ml

[0256] ALKO4237: 650 ECU/ml

[0257] Purified proteins:

[0258] 20K-cellulase: 9423 ECU/ml

[0259] 50K-cellulase: 10100 ECU/ml

[0260] The results are shown in Tables XI-XIII.

[0261] ALKO4179, ALKO4265 and ALKO4237 cellulase preparations and 20K-and 50K-cellulases stay almost 100% stable for 30 minutes at 40° C. inall three tested detergents. ALKO4125 stays stable for 30 minutes at 40°C. in Colour Detergent Liquid and in OMO® Neste. TABLE XI Stability ofdifferent cellulases in 0.25% Colour Detergent Liquid (pH 7.5-7.9).enzyme dosage % of activity left preparation % (ml) pH* 0′ 5′ 10′ 20′30′ Culture filtrates: ALKO4125 6 7.3 100 97 98 98 99 ALKO4179 6 7.1 10099 100 100 10 ALKO4265 6 7.2 100 100 100 100 100 ALKO4237 6 7.1 100 10082 95 100 Purified proteins from ALKO4237: 20K-cellulase 1 7.8 100 98 9997 100 50K-cellulase 1 7.6 100 100 100 100 100

[0262] TABLE XII Stability of different cellulases in 0.25% OMO ® Total(or OMO ® Neste pH 8.5). enzyme dosage % of activity left preparation %(ml) pH* 0′ 5′ 10′ 20′ 30′ Culture filtrates: ALKO4125 6 7.8 100 98 9686 87 ALKO4179 6 7.3 100 98 96 96 99 ALKO4265 6 7.1 100 100 100 100 100ALKO4265 4 7.8 100 99 97 100 100 ALKO4237 4 7.8 100 100 100 99 100ALKO4237 2 7.3 100 99 97 99 99 Purified proteins from ALKO4237:20K-cellulase 1 8.2 100 100 99 93 100 50K-cellulase 1 7.8 100 95 92 9594

[0263] TABLE XIII Stability of different cellulases in 0.25% OMO ® Color(pH 9.6-10) enzyme dosage % of activity left preparation % (ml) pH* 0′5′ 10′ 20′ 30′ Culture filtrates: ALKO4125 6 9.6 100 (15)  (15)  (13) (14)  ALKO4179 6 8.3 100 97 100 97 99 ALKO4265 6 9.1 100 100 100 100 100ALKO4265 4 8.5 100 93 95 99 98 ALKO4237 4 8.5 100 98 96 96 99 ALKO4237 29.1 100 93 95 99 98 Purified proteins from ALKO4237: 20K-cellulase 1 9.8100 99 100 100 100 50K-cellulase 1 8.9 100 100 100 100 100

Example 14

[0264] Function of Neutral Cellulases in Detergents in HEC Substrate

[0265] The function of different neutral cellulases in detergents wasdetermined by using hydroxyethylcellulose (HEC) as a substrate. Thetested cellulase preparations were ALKO4265 and ALKO4237 concentratedculture filtrates and purified 20K- and 50K-cellulases from ALKO4237strain. HEC substrates were prepared by dissolving 1% HEC into 0.25%detergent solutions. By using these substrates the activity against HEC(ECU/ml) was measured at 40° C. from each cellulase preparations asdescribed in Example 1. Detergents and cellulase preparations used inthese experiments are described in Example 13.

[0266] pH of the substrates:

[0267] HEC/buffer pH 7

[0268] HEC/Colour Detergent Liquid pH 7.5

[0269] HEC/OMO® Total pH 7.8

[0270] HEC/OMO® Color pH 9.7 TABLE XIV ECU of the cellulase preparationsin different detergents (compared as % from the ECU activity measured inpH 7 buffer) Activity % ECU/ ECU/ ECU/ ECU/ preparation buffercol.det.liquid OMO ® Total OMO ® Color culture filtrates: ALKO4265 10089 96 59 ALKO4237 100 97 95 40 purified proteins: 20K-cellulase 100 10093 81 50K-cellulase 100 92 79 46

[0271] ALKO4237 and ALKO4265 cellulase preparations and 20K- and50K-cellulases function in all three tested detergents when using HEC asa substrate.

Example 15

[0272] Use of Neutral Cellulases in detergents on Cotton Woven Fabrics

[0273] In this experiment is described the ability of the neutralcellulases to function as fabric-softening agent and to prevent fuzzingand thus to reduce pilling tendency from cotton fabric after repeatedlaunderings in detergents. The tested cellulase preparations wereALKO4237 concentrated culture filtrate and the purified 20K- and50K-cellulases from ALKO4237 strain (Examples 1 and 9).

[0274] The washing experiment was carried out with a Launder-Ometer LP-2(Atlas, Ill., USA). About 10 g of prewashed (Example 3) unbleachedcotton woven fabric swatch was loaded into 1.2 liter container thatcontained 150 ml of 0.25% detergent solution with or without cellulase.Cellulase dosages were based on protein amounts. Detergent solutionswere OMO® Total (Lever, UK) and Colour Detergent Liquid (Unilever, TheNetherlands). A quantity of steel balls were added into each containerto increase the mechanical action. The Launder-Ometer was run at 42 rpmfor 0.5 or 1 hour at 40° C. The material was washed 4 times withintermediate rinsing and drying.

[0275] Weight loss (see Example 0.6) was used to decribe the amount offuzz removed from the fabrics surface. TABLE XV Weight loss of thefabrics after the first washing time with neutral cellulases indetergents. enzyme dosage weight sample as protein/ time loss nopreparation g fabric h % In Colour Detergent Liquid: 1 — — 1 0.05 2ALKO4237 11 1 0.3 3 ALKO4237 22 1 0.7 4 20K-cellulase 2 1 0.1 520K-cellulase 5 1 0.5 6 20K-cellulase 8 1 1.0 7 50K-cellulase 2 1 0.1 850K-cellulase 5 1 0.2 9 — — 0.5 0.2 10 20K-cellulase 8 0.5 0.5 In OMO ®Total: 11 — — 1 0.03 12 20K-cellulase 8 1 1.1 13 — — 0.5 0.1 1420K-cellulase 8 0.5 0.7

[0276] In the Table XV it is shown that after the first washing inLaunder-Ometer weight loss of the fabrics were increased clearly morewith cellulase treated fabrics than with the fabrics treated with thesole detergent Also weight loss was increased as a function of cellulasedosage and further with 20K-cellulase weight loss was increased whenwashing time was raised from 0.5 hour to 1 hour. 20K-cellulase workedequally well in Colour Detergent Liquid and in OMO® Total. These resultsindicate that particularly the 20K-cellulase and ALKO4237 cellulasepreparation function in detergents as fuzz removing agents after alreadyone wash time.

[0277] After three further washing times with samples 1, 2, 4 and 7(Table XV) the evaluation of the fabrics was performed by a panelconsisting of three persons. Panelists were asked to evaluate thesoftness and visual appearance of the treated fabrics as follows.

[0278] The Softness of the fabrics:

[0279] A. the fabric treated with cellulase is softer than the fabrictreated without cellulase

[0280] B. the fabric treated with cellulase is as soft as the fabrictreated without cellulase

[0281] C. The fabric treated with cellulase is harder than the fabrictreated without cellulase

[0282] The results are shown in Table XVI.

[0283] Visual appearance of the fabrics was evaluated by ranking thefabrics on a score from 1 to 5. Score of 5 gave no fuzz or pills and thefabric texture became more apparent. Score of 1 gave many pills andfuzz. Total score for each fabric was calculated and divided by thenumber of the panelists. The average score of the visual appearance ofeach fabric is shown in Table XVI. TABLE XVI Softness and visualappearance of the fabrics after 4 repeated washing times with neutralcellulases in detergents. enzyme dosage as protein/ time visualpreparation g fabric h softness appearance In Colour Detergent Liquid: —— 1 1 ALKO4237 11 1 100%: softer 3.2 with cellulase 20K- 2 1 100%:softer 3.7 cellulase with cellulase 50K- 2 1 100%: no 1.7 cellulasedifference

[0284] After the 4 treatments the cellulase treated fabrics had clearlybetter visual appearance than the fabrics that were treated with soledetergent. Thus fabrics treated with these cellulases maintained goodappearance and the fuzziness was prevented after repeated washingscompared to the fabric treated without cellulases. Also after 4 washtimes the ALKO4237 and 20K-cellulase treated fabrics were softer thanthe fabric treated with sole detergent.

Example 16

[0285] Use of Neutral Cellulases in Detergents on Cotton Fleecy Knit

[0286] In this experiment is described the ability of the neutralcellulases to function as fabric-softening agent and to prevent fuzzingand thus to reduce pilling tendency from coloured cotton fleecy knitafter repeated launderings in detergents. The tested cellulasepreparations were ALKO4237 concentrated culture filtrate and thepurified 20K-cellulase from ALKO4237 strain (Examples 1 and 9).

[0287] Green cotton fleecy knit swatches were washed at Launder-Ometerin Colour Liquid Detergent or in OMO® Total for 1 h 3 or 10 times withor without cellulases as described in Example 15.

[0288] The evaluation of the knits was performed by a panel consistingof three persons. Panelists were asked to evaluate the softness andvisual appearance (both right and reverse sides) of the treated knits asdescribed in Example 15. Weight loss of the knits was determined asdescribed in Example 15. The results are shown in Table XVII.

[0289] After the 3 washing times the 20K-cellulase treated knits hadbetter visual appearance both on the right and reverse side than theknits treated with sole detergent. Knits treated 10 times with ALKO4237cellulase preparation had clearly better visual appearance and brightergreen colour than the knits treated only with detergent. The bettervisual appearance of the cellulase treated knits was detected alreadyafter 1 wash time (especially on the reverse side) and it was furtherdeveloped during the additional washings. The cellulase treated knitswere also softer than the knits treated with sole detergent. TABLE XVIISoftness, weight loss and visual appearance of the fleecy knits after 3or 10 repeated washing times with or without cellulases in detergents.Before washings pH of the 0.25% Colour Detergent Solution was 7.9 and8.4 of the 0.25% OMO ® Total solution. enzyme pH weight appearancedosage as washing after loss visual reverse preparation protein/g fabrictimes washings % softness right side Colour Detergent Liquid — — 3 7.90.46 1 1 20K* 5 3 7.4 0.88  33%: softer with cellulase 1.5 2.7 — — 108.0 1.46 1 1 A4237 20 10 7.9 2.80 100%: softer with cellulase 2.5 2.8OMO ® Total — — 10 8.3 0.57 1 1 A4237 20 10 8.2 1.57 100%: softer withcellulase 2.3 2.8

Example 17

[0290] Use of Neutral Cellulases in Detergents on Aged Cotton FleecyKnit

[0291] In this experiment is described the ability of the neutralcellulases to function as fabric-renewal and -softening agent.

[0292] Green cotton fleecy knit was washed 10 times, with intermediatedrying, in Cylinda washing machine with programme 3 at 60° C., 10 ml ofOMO® Color (Lever, UK). This was to simulate the washings of the knit inpractice.

[0293] After 10 treatments this aged knit had unattractive and fadedappearance with a lot of fuzz at the surface.

[0294] After these 10 repeated washes the fleecy knit was used for thewashing experiments with or without cellulase. Knit swatches were washedat Launder-Ometer in Colour Liquid Detergent for 1 h 1 to 3 times asdescribed in example 15 with intermediate rinsing and drying. Thecellulase preparations used were ALKO4237 concentrated culture filtrateand purified 20K- and 50K-cellulases from ALKO4237 (Example 9).

[0295] The evaluation of the knits was performed by a panel consistingof three persons. Panelists were asked to evaluate the softness andvisual appearance (both right and reverse sides) of the treated knits asdescribed in Example 15. Weight loss of the knits was determined asdescribed in Example 15. The results are shown in Table XVIII.

[0296] After one wash time ALKO4237 and 20K-cellulase treated knits hadslightly better visual appearance than the knit treated with soledetergent. The good visual appearance and more attractive look wasfurther developed to the 20K-cellulase treated knits after 2 and 3 washtimes. Visual appearance was also improved after two wash times on theknits treated with 50K-cellulase compared to the knit treated with soledetergent. As general, the knits treated with cellulases had clearlyimproved and attractive look while the knits treated without cellulasehad still unattractive and faded appearance. TABLE XVIII Softness,weight loss and visual appearance of the aged fleecy knits after 1 to 3repeated washing times with or without cellulases in detergents. Beforewashings pH of the 0.25% Colour Detergent Solution was 7.9. enzymevisual dosage as pH appearance mg protein/ washing after weight rightpreparation g fabric times washings loss % softness side reverse — — 1ND 0 1 1 ALKO4237 20  1 ND 0.61 100%: no difference 1 1.5 20K* 5 1 ND 0100%. no difference 1.5 1.5 — — 2 7.9 0.10 1 1 20K* 5 2 7.7 0.46 100%:softer with cellulase 2.5 2.2 50K* 5 2 7.7 0.26 100%: no difference 11.2 50K* 15  2 7.3 0.49 100%: no difference 1 1.3 — — 3 ND 0.31 1 1 20K*5 3 ND 0.88 100%: softer with cellulase 3.0 2.2

Example 18

[0297] Isolation of the ALKO4237 Chromosomal DNA and Construction of theGenomic Library

[0298]Melanocarpus albomyces ALKO4237 was grown in shake flask culturesin potato dextrose (PD; Difco, USA)-medium at 42° C., 250 rpm for 3days. The chromosomal DNA was isolated according to Raeder and Broda,Lett. Appl. Microbiol. 1:17-20 (1985). Briefly, the mycelium was washedwith 20 mM EDTA and lysed in extraction buffer (200 mM Tris-HCl (pH8.5), 250 mM NaCl, 25 mM EDTA, 0.5% SDS). The DNA was extracted withphenol and a mixture of chloroform:isoamyl alcohol (24:1 v/v). RNA wasdigested with RNase.

[0299] The chromosomal DNA was partially digested with Sau3A (BoehringerMannheim, Germany) and treated with calf intestine alkaline phosphatase.DNA ranging from 5-15 kb was isolated from an agarose gel usingbeta-agarase (Boehringer Mannheim, Germany) and used to construct thegenomic ALKO4237 library.

[0300] The predigested Lambda DASH®II BamHI Vector Kit (Stratagene, USA)was used to construct the library and the instructions of themanufacturer were followed in all the subsequent steps. Briefly, about200 ng of the size-fractionated DNA was ligated into 1 μg of DASH®IIprepared arms, and packaged using Gigapack II packaging extract(Stratagene, USA). The titer of the library was determined by infectingE. coli XL 1-Blue MRA (P2)-cells with serial dilutions of the packagedphage and plating on NZY plates. The library was stored at 4° C. inSM-buffer, with 4% (v/v) chloroform. It was used for screening withoutamplification.

Example 19

[0301] Amplification, Cloning and Sequencing of the 20K-cellulase DNAwith Degenerate Primers

[0302] To amplify the 20K-cellulase gene by polymerase chain reaction(PCR), a pair of degenerate primers based on the peptide sequences (FIG.17) was synthesized. Primer 1 (429-32) was derived from the amino acids#8-14 of the N-terminal peptide #429 (FIG. 17), and primer 2 (fr28-16)was designed as the antisense strand for the amino acids #2-8 of thepeptide fr28 (FIG. 17). Additional EcoR1 restriction sites were added atthe 5′-termini to facilitate the cloning of the amplified fragment.Primer 1 (429-32)        EcoRI 5′-ATA GAATTC TA(C/T) TGG GA(C/T) TG(C/T)TG(C/T) AA(A/G) CC               Y       W   D       C       C       K       P Primer 2(fr28-16)        EcoRI 5′-ATA GAATTC TT (A/G)TC (A/C/G/T)GC (A/G)TT(C/T)TG (A/G)AA CCA              N       D           A       N       Q       F   W

[0303] In the PCR reaction, 1 μg of the purified ALKO4237 genomic DNA(Example 18) was used as the template. Dynazyme DNA polymerase(Finnzymes Ltd, Finland) was used according to the supplier'sinstructions. Template DNA (0.7 μg/μl) 1.4 μl Primer 1 (0.5 μg/μl) 1 μlPrimer 2 (0.5 μg/μl) 1 μl dNTPs (2 mM) 5 μl 10 × PCR buffer 10 μl dH2O82 μl Dynazyme (2 U/μl) 1 μl Total 101.4 μl

[0304] The PCR reaction was performed under the following conditions:Step 1 95° C. 5 min Step 2 95° C. 1 min Step 3 56° C. 1 min Step 4 72°C. 1 min Step 5 go to “step 2” 29 more times Step 6 72° C. 8 min Step 7 4° C. hold

[0305] Ten μl of reaction mixture was analyzed by agarose gelelectrophoresis, and a single band corresponding to about 600 bp inlength was detected. The remaining of the PCR product was digested withEcoR1 restriction endoglucanase, and run by agarose electrophoresis. Theagarose section containing the DNA fragment was excised, and purified bythe Magic PCR Preps (Promega, USA) method according to supplier'sinstructions. The isolated fragment was ligated with pBluescript II SK+(Stratagene, USA) plasmid which was cut similarly with EcoR1. CompetentEscherichia coli XL-Blue cells (Stratagene, USA) were transformed withthe ligation mixture. Plasmid DNA from a few of the resulting colonieswas isolated by the Magic Minipreps (Promega, USA) method according tosupplier's instructions. The plasmid DNA was analyzed by agaroseelectrophoresis, and one clone with expected characteristics wasdesignated pALK549.

[0306] The Melanocarpus DNA from pALK549 was sequenced by using ABI(Applied Biosystems, USA) kits based on fluorescent-labeled T3 and T7primers, or sequence-specific primers with fluorescent-labeleddideoxynucleotides by the Taq dye primer cycle sequencing protocol inaccordance with the supplier's instructions. Because of high GC contentof the Melanocarpus DNA, the sequencing reactions were performed atannealing temperature of 58° C., with 5% (v/v) DMSO. Sequencingreactions were analyzed on ABI 373A sequencer (Applied Biosystems, USA),and the sequences obtained were characterized by using the GeneticsComputer Group Sequence Analysis Software Package, version 7.2.

[0307] The insert (594 bp) in pALK549 was found to encode the majorityof the 20K-cellulase derived peptides (FIG. 17). The PCR amplified DNA(in addition to the primers) corresponds to the nucleotides 175-716 inFIG. 19.

[0308] Chromosomal DNA from Myriococcum sp. ALKO4124 was isolated asdescribed in Example 18. A PCR reaction with the primers 429-32 andfr28-16 and ALKO4124 chromosomal DNA as the template produced a fragmentof same size as from ALKO4237 DNA. This fragment was partly sequenced,and was almost identical to the ALKO4237 sequence. It is concluded thatMyriococcum sp. ALKO4124 has a protein, which is almost identical to the20K-cellulase of Melanocarpus albomyces ALKO4237. This result is also inagreement with the observation that the ALKO4237 20K-cellulase specificantibodies also recognize a 20K protein band from ALKO4124 growth mediumin Western analysis (FIG. 14). Enzymes from both strains gave similargood results in biostoning experiments (Examples 3 and 4).

Example 20

[0309] Cloning and Sequencing the Melanocarpus albomyces ALKO423720K-Cellulase Gene

[0310]E. coli XL 1-Blue MRA (P2)-cells (Stratagene, USA) were grown inLB+0.2% maltose+10 mM MgSO₄, and diluted to OD₆₀₀=0.5. The cells wereinfected with the Melanocarpus albomyces ALKO4237 genomic library(Example 18) for 15 min at 37° C., and plated with NZY top agar on theNZY plates. Plates were incubated at 37° C. overnight. The plaques weretransferred onto a nylon filter (Hybond, Amersham, UK) according toStratagene's instructions.

[0311] The purified PCR fragment (Example 19) was labeled withdigoxigenin according to Boehringer, DIG DNA Labeling and DetectionNonradioactive, Application Manual. Hybridization was performed at 68°C. The positive clones were picked in SM buffer/chloroform, and purifiedwith a second round of screening.

[0312] Under these conditions 4 positive clones were found. The largescale bacteriophage lambda DNA isolation from the clones was doneaccording to Sambrook et al., in Molecular Cloning: A Laboratory Manual,2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. The phage DNAs were analyzed by digestion of the DNA withseveral restriction enzymes, and the digested DNA was hybridized withthe PCR-probe. Three hybridizing fragments were isolated: about 2.6 kbEcoR1-XhoI fragment, about 4.9 kb XhoI fragment and about 3 kb SacIfragment. These were inserted into similarly cut pBluescript II SK+vector (Stratagene, USA), creating plasmids pALK1221, pALK1222 andpALK1223, respectively (FIG. 18).

[0313] The Melanocarpus albomyces DNA in pALK1221 was sequenced asdescribed in Example 19. The DNA sequence encoding the Melanocarpusalbomyces 20K-cellulase is shown in FIG. 19. The sequence is 936 bp inlength, and has an open reading frame (ORF) coding for 235 amino acids;the gene has two introns. The putative signal peptide processing site isafter alanine-21, and the N-terminus of the mature protein begins atalanine-22, as suggested by the peptide sequencing results (FIG. 17,peptide #429). The ORF predicts a protein with a molecular weight of25.0 kDa for the full-length preprotein, and 22.9 kDa for the matureprotein. This is in good agreement with the results obtained from theprotein purification work (Example 10). These results also verify thatthe about 35 kDa protein detected previously with the 20K-cellulaseantiserum (Example 10) is a different gene product than the20K-cellulase.

[0314] The 20K-cellulase of Melanocarpus albomyces appears to belong tofamily K of cellulases and family 45 of glycosyl hydrolases (Henrissat &Bairoch, Biochem. J. 293:781-788 (1993)). The 20K-cellulase showshomology (about 76% identify in 235 amino acid overlap) towards theHumicola insolens endoglucanase V (embl:a23635), but the 20K-cellulasehas the surprising feature that it does not harbor the cellulose bindingdomain (CBD) and its linker, which are characteristic of the Humicolainsolens endoglucanase V and other related endoglucanases (Schülein etal., 1993, In: Suominen & Reinikainen (eds), Foundation for Biotechnicaland Industrial Fermentation Research, Helsinki, vol. 8, 109.; Saloheimoet al., 1994, Mol. Microbiol. 13, 219). This feature of the20K-cellulase may account for the excellent performance of the enzyme inbiostoning experiments (Example 10).

Example 21

[0315] Amplification, Cloning and Sequencing of 50 K-Cellulase DNA withDegenerate Primers

[0316] The peptides derived from the 50K-cellulase (Table IX) sharedsome homology towards Humicola grisea endoglucanase I (DDBJ:D63516). Toamplify the 50 K-cellulase gene by polymerase chain reaction (PCR) apair of degenerate primers based on the peptide sequences (Table IX) wassynthetized Primer 1 (507-128) was derived from the amino acids #5-10 ofthe peptide #507 (Table IX), and primer 2 (509-rev) was designed as theantisense strand for the amino acids #4-9 of the peptide 509 (Table IX).The order of the two peptides in the protein—and the correspondingsense-antisence nature of the primers—was deduced from comparison withthe Humicola grisea endoglucanase I. Primer 1 (507-128) 5′-GA(C/T)GA(A/G) AC(A/C/G/T) GA(A/G) CA(C/T) (A/C)G   D        E       T           E       H       R Primer 2 (509-rev)5′-TA (A/C/G/T)GC (A/C/G/T)CC (A/C/G/T)CC (A/C/G/T)GG (A/G)TT    Y          A            G           G           P       N

[0317] In the PCR reaction, 1.5 μg of the purified ALKO4237 genomic DNA(Example 18) was used as the templete. Dynazyme DNA polymerase(Finnzymes Ltd, Finland) was used according to the supplier'sinstructions. Template DNA (0.3 μg/μl) 5 μl Primer 1 (0.5 μg/μl) 1 μlPrimer 2 (0.5 μg/μl) 1 μl dNTPs (2 mM) 5 μl 10 × PCR buffer 10 μl dH2O79 μl Dynazyme (2 U/μl) 1 μl Total 102 μl

[0318] The PCR reaction was performed under the following conditions:Step 1 95° C. 5 min Step 2 95° C. 1 min Step 3 56° C. 1 min Step 4 72°C. 1 min Step 5 go to “step 2” 29 more times Step 6 72° C. 8 min Step 7 4° C. hold

[0319] Ten μl of reaction mixture was analyzed by agarose gelelectrophoresis, and a single band corresponding to about 160 bp inlength was detected. The remaining of the PCR product was loaded on aagarose gel electrophoresed, and the agarose section containing the DNAfragment was excised, and purified by the Magic PCR Preps (Promega, USA)method according to the supplier's instructions.

[0320] The isolated fragment was ligated with pBluescript II SK+(Stratagene, USA) plasmid which had been digested with EcoRVendonuclease, and ddT-tailed as described in Holton and Graham (1990)Nucl. Acids Res. 19, 1156. Competent Escherichia coli XL-Blue cells(Stratagene, USA) were transformed with the ligation mixture. PlasmidDNA from a few of the resulting colonies was isolated by the MagicMinipreps (Promega, USA) method according to the supplier'sinstructions. The plasmid DNA was analyzed by agarose electrophoresis,and one clone with expected characteristics was designated pALK1064.

[0321] The insert (161 bp) in pALK1064 was sequenced as described inExample 19, and was found to contain an ORF, which predicted a peptidehomologous to Humicola grisea endoglucanase I (DDBJ:D63516). The ORFalso encoded the peptide #612 (Table IX) from the purified50K-cellulase. The PCR amplified DNA (in addition to the primers)corresponds to the nucleotides 404-530 in FIG. 21.

[0322] PCR with the primers 507 and 590-rev with ALKO4124 chromosomalDNA as template (Example 19) produced a fragment of same size as fromALKO4237 DNA. This suggests that Myriococcum sp. ALKO4124 has a proteinvery similar to the 50K-cellulase of Melanocarpus albomyces ALKO4237.This is also supported by the fact that enzymes from both strains gavesimilar good results in biostoning experiments.

Example 22

[0323] Cloning and Sequencing the Melanocarpus albomyces ALKO423750K-Cellulase Gene

[0324] The genomic bank of Melanocarpus albomyces ALKO4237 was preparedfor hybridization as described in Example 20. The purified PCR fragmentcarrying part of the 50K-cellulase gene (Example 21) was labeled withdigoxigenin according to Boehringer, DIG DNA Labeling and DetectionNonradioactive, Application Manual. Hybridization was performed at 68°C. The positive clones were picked in SM buffer/chloroform, and purifiedwith a second round of screening.

[0325] Under these conditions 10 positive clones were found. The largescale bacteriophage lambda DNA isolation from the clones was doneaccording to Sambrook et al., 1989. The phage DNAs were analyzed bydigestion of the DNA with several restriction enzymes, and the digestedDNA was hybridized with the 50K-cellulase-specific PCR-probe. Fourhybridizing fragments were isolated: about 2.8 kb SacI-XhoI fragment,about 5 kb SacI fragment, about 3.2 kb XhoI fragment, and about 2 kbEcoRI fragment. These were inserted into similarly cut pBluescript IISK+ vector (Stratagene, USA), creating plasmids pALK1234, pALK1233,pALK1226 and pALK1227, respectively (FIG. 20).

[0326] The Melanocarpus albomyces ALKO4237 DNA was sequenced from the50K-cellulase specific plasmids mentioned above. The sequencing protocolhas been described in Example 19.

[0327] The DNA encoding the Melanocarpus albomyces 50K-cellulase isshown in FIG. 21 (A and B). The sequence reveals an ORF of about 1363 bpin length, interrupted by one intron. The ORF codes for 428 amino acids.The predicted protein has a molecular weight of 46.8 kDa and aftersignal peptide cleavage of 44.8 kDa. All the peptides in Table IX arefound in the predicted protein sequence (FIG. 2), although some aminoacids identified with uncertainty during the peptide sequencing provedto be incorrect. The protein shows homology to Humicola griseaendoglucanase I (DDBJ:D63516).

Example 23

[0328] Amplification, Cloning and Sequencing of 50K-Cellulase B DNA withDegenerate Primers

[0329] The peptides derived from the 50K-cellulase B (Table X) sharedsome homology towards Humicola grisea cellobiohydrolase I (DDBJ:D63515).To amplify the 50K-cellulase B gene by polymerase chain reaction (PCR) apair of degenerate primers based on the peptide sequences (Table X) wassynthesized. Primer 1 (636) was derived from the amino acids #1-5 of thepeptide #636 (Table X) (the first amino acid was guessed to be lysine,because this peptide was isolated after digestion with a proteasecleaving after lysines), and primer 2 (534-rev) was designed as theantisense strand for the amino acids #3-8 of the peptide #534 (Table X).The order of the two peptides in the protein—and the correspondingsense-antisense nature of the primers—was deduced from comparison withthe Humicola grisea cellobiohydrolase I. Primer 1 (636) 5′-AA(A/G)CA(C/T) GA(A/G) TA(C/T) GG(A/C/G/T) AC    K      H       E       Y       G           T Primer 2 (534-rev)5′-CC (A/G)TA (A/G)AA (A/G)TC (A/C/G/T) GG (A/G)TT    G       Y       F       D            P       N

[0330] In the PCR reaction, 1.5 μg of the purified ALKO4237 genomic DNA(Example 18) was used as the template. Dynazyme DNA polymerase(Finnzymes Ltd, Finland) was used according to the supplier'sinstructions. Template DNA (0.3 μg/μl) 5 μl Primer 1 (0.3 μg/μl) 1.7 μlPrimer 2 (0.3 μg/μl) 1.7 μl dNTPs (2 mM) 5 μl 10 × PCR buffer 10 μl dH2O80 μl Dynazyme (2 U/μl) 1 μl Total 104.4 μl

[0331] The PCR reaction was performed under the following conditions:Step 1 95° C. 5 min Step 2 95° C. 1 min Step 3 48° C. 1 min Step 4 72°C. 2 min Step 5 go to “step 2” 34 more times Step 6 72° C. 8 min Step 7 4° C. hold

[0332] Twenty μl of reaction mixture was analyzed by agarose gelelectrophoresis, and a few bands were detected. One of the bands had anapparent size of 700 bp, which size was in agreement with size one wouldexpect, when comparing with Humicola grisea cellobiohydrolase gene,particularly, if the fragment contained one or more introns. The PCRproducts were purified by the Magic PCR Preps (Promega, USA) methodaccording to the supplier's instructions.

[0333] The isolated fragments was ligated with pBluescript II SK+(Stratagene, USA) plasmid which had been digested with EcoRVendonuclease, and ddT-tailed as described in Holton and Graham, Nucl.Acids Res. 19:1156 (1990). Competent Escherichia coli XL-Blue cells(Stratagene, USA) were transformed with the ligation mixture. PlasmidDNA from a few of the resulting colonies was isolated by the MagicMinipreps (Promega, USA) method according to the supplier'sinstructions. The plasmid DNA was analyzed by agarose electrophoresis,and one clone with about 700 bp insert was designated pALK1224.

[0334] The insert in pALK 1224 was sequenced as described in Example 19,and was found to contain an ORF encoding the whole peptide #636 from the50K-cellulase B (Table X). The ORF predicted a peptide homologous toHumicola grisea cellobiohydrolase I (DDBJ:D63515). The PCR amplified DNA(in addition to the primers) corresponds to the nucleotides 371-1023 inFIG. 23.

Example 24

[0335] Cloning and Sequencing the Melanocarpus albomyces ALKO423750K-Cellulase B Gene

[0336] The genomic bank of Melanocarpus albomyces ALKO4237 was preparedfor hybridization as described in Example 20. The insert in pALK1224 wasremoved by digesting the plasmid with restriction endoglucanases EcoRIand HindIII. The digested plasmid DNA was run by agaroseelectrophoresis. The agarose section containing the about 700 bp DNAfragment was excised, and purified by the Magic PCR Preps (Promega, USA)method according to the supplier's instructions.

[0337] The purified PCR fragment from pALK1224 carrying part of the50K-cellulase B gene (Example 23) was labeled with digoxigenin accordingto Boehringer, DIG DNA Labeling and Detection Nonradioactive,Application Manual. Hybridization was performed at 68° C. The positiveclones were picked in SM buffer/chloroform, and purified with a secondround of screening.

[0338] Under these conditions 3 positive clones were found. The largescale bacteriophage lambda DNA isolation from the clones was doneaccording to Sambrook et al., in Molecular Cloning: A Laboratory Manual,2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. The phage DNAs were analyzed by digestion of the DNA withseveral restriction enzymes, and the digested DNA was hybridized withthe 50K-cellulase B specific PCR probe. A hybridizing 3.5 kb NotIfragment was isolated, and inserted into similarly cut pBluescript IISK+ vector (Stratagene, USA), creating plasmid pALK1229 (FIG. 22).

[0339] The extreme 5′-end of the gene was found by hybridizing the phageDNAs with 0.2 kb NotI-PstI-fragment from pALK1229. A hybridizing 2.4 kbPstI-fragment was isolated and inserted into similarly cut pBluescriptII SK+ vector (Stratagene, USA), creating plasmid pALK1236 (FIG. 22).

[0340] Part of the inserts in pALK1229 and pALK1236 were sequenced asdescribed in Example 19. The DNA encoding the Melanocarpus albomyces50K-cellulase B is shown in FIG. 23 (A and B). The sequence reveals anORF of 1734 bp in length interrupted by five introns. The ORF codes for452 amino acids. The predicted protein has a molecular weight of 49.9kDa and after signal peptide cleavage of 47.6 kDa. All the peptides inTable X are found in the predicted protein sequence (FIGS. 23A and B),although some amino acids identified with uncertainty during the peptidesequencing proved to be incorrect. The predicted protein shows homologyto Humicola grisea cellobiohydrolase I (DDBJ:D63515) and othercellobiohydrolases. However, 50K-cellulase B has the surprising featurethat it does not harbor the cellulose binding domain (CBD) and itslinker, which is characteristic to Humicola grisea cellobiohydrolase Iand many other cellobiohydrolases.

Example 25

[0341] Screening the Melanocarpus albomyces ALKO4237 Genomic Librarywith Trichoderma reesei Cellulases Genes

[0342] The genomic bank of Melanocarpus albomyces ALKO4237 was preparedfor hybridization as described in Example 20.

[0343] A DNA fragment carrying Trichoderma reesei cbh1 specific DNA wasisolated by cutting plasmid pTTc01 (FIG. 24) with restrictionendonuclease HincII, and isolating the about 1.6 kb fragment fromagarose gel after electrophoresis. A DNA fragment carrying Trichodermareesei egl2 specific DNA was isolated by cutting plasmid pMS2 (FIG. 25)with restriction endonucleases BamHI and EcoRI, and isolating the about1.5 kb fragment from agarose gel after electrophoresis. The cloning ofthe cbh1 gene is described in Teeri et al., Bio/Technology 1:696-699(1983) and the DNA sequence is described in Shoemaker et al.,Bio/Technology 1: 691-696 (1983). The egl2 (originally called “egl3”)gene is described in Saloheimo et al., Gene 63:11-21 (1988).

[0344] The fragments were labeled with digoxigenin according toBoehringer, DIG DNA Labeling and Detection Nonradioactive, ApplicationManual. Hybridization was performed at 68° C. with the cbh1 probe and at60° C. with the egl2 probe. The positive clones were picked in SMbuffer/chloroform, and purified with a second round of screening.

[0345] Under these conditions 13 cbh1 positive and 6 egl2 positiveclones were found. One clone hybridized to both probes. The lambda DNAwas isolated from the clones as described above. The phage DNAs wereanalyzed by digestion of the DNA with several restriction enzymes, andthe digested DNA was hybridized with the cbh1 and egl2 probes. Theclones were also hybridized with the 20K-cellulase-specific PCR fragment(Example 19). One clone (lambda-16) was clearly positive, and two otherclones (lambda-8/1 and lambda-5/2) were weakly positive; all theseclones were originally picked with the cbh1 probe.

[0346] An about 4 kb EcoRI fragment from lambda-16, which hybridized toboth the Trichoderma reesei cbh1 probe and to the 20K-cellulase specificPCR fragment, was isolated from agarose gel after electrophoresis, andinserted into similarly cut pBluescript II SK+. The resulting plasmidwas named pALK1230 (FIG. 26).

[0347] Part of the insert in pALK1230 was sequenced as described inExample 19. The DNA appears not to encode the 20K-cellulase, but codesfor a protein homologous to several cellulases, particularly at thecellulose binding domain (CBD) area. Thus the gene product very likelyhas high affinity towards cellulosic material, and therefor this geneproduct was designated as protein-with-CBD. The sequence is shown inFIG. 27.

[0348] PCR reactions with the primers 636 and 534-rev (Example 23) wereperformed with the DNA from the 19 lambda clones as templates. Onelambda clone, lambda-3, gave a band about 700 bp in size, similar tothat in Example 23 when ALKO4237 chromosomal DNA was used as a template.This clone had originally been picked by the Trichoderma cbh1 probe. Thelambda DNA was digested with several restriction endonucleases, andhybridized with the 50K-cellulase B specific probe. The clone showedsimilar restriction enzyme pattern as the 3 clones in Example 24. It isconcluded that lambda-3 also carries the 50K-cellulase B gene.

Example 26 Fusion Proteins

[0349] A recombinant vector encoding the 20K-cellulase, 50K-cellulase orthe 50K-cellulase B is prepared by fusing the cellulase encodingsequence with the sequence of Trichoderma reesei cellulase orhemicellulase or at least one functional domain of said cellulase orhemicellulase, as described in U.S. Pat. No. 5,298,405, WO 93/24621 andin Genbank submission L25310, incorporated herein by reference.Especially, the enzyme is selected from the group consisting of CBHI,CBHII, EGI, EGII, XYLI, XYLII and MANI, or a domain thereof, such as thesecretion signal or the core sequence.

[0350] Fusion proteins can be constructed that contain an N-terminalmannanase or cellobiohydrolase or endoglucanase core domain or the coreand the hinge domains from the same, fused to one of the Melanocarpuscellulase sequences. The result is a protein that contains an N-terminalmannanase or cellobiohydrolase or endoglucanase core or core and hingeregions, and a C-terminal Melanocarpus cellulase. The fusion proteincontains both the Trichoderma mannanase or cellobiohydrolase orendoglucanase and the Melanocarpus cellulase activities of the variousdomains as provided in the fusion construct. Alternatively, mutationsthat modify the activities of the Trichoderma mannanase orcellobiohydrolase or endoglucanase, or the Melanocarpus cellulaseactivities, may be included in the constructions. In this case, thefusion proteins contain both the modified Trichoderma enzyme activityand the Melanocarpus cellulase activity of the various domains asprovided in the fusion construct.

[0351] Fusion proteins can also be constructed such that the mannanaseor cellobiohydrolase or endoglucanase tail or a desired fragmentthereof, is placed before one of the Melanocarpus cellulase sequences,especially so as to allow use of a nonspecific protease site in the tailas protease site for the recovery of the Melanocarpus cellulase partfrom the expressed fusion protein. Alternatively, ftision proteins canbe constructed that provide for a protease site in a synthetic linkerthat is placed before one of the Melanocarpus cellulases, with orwithout the tail sequences.

Example 27 Hosts

[0352] The recombinant construct encoding the desired fusion proteins orMelanocarpus proteins are prepared as above, and transformed into afilamentous fungus such as Aspergillus spp., preferably Trichoderma spp.

Example 28 Trichoderma Background for 20K-Cellulase Production

[0353] In this example is described stone-washing experiments todetermine the most suitable background of Trichoderma cellulases for20K-cellulase production. The purpose of these experiments was todetermine which Trichoderma cellulases would cause backstaining instone-washing at neutral conditions.

[0354]Trichoderma reesei strain ALKO3620 (endoglucanase 2 gene isdeleted) was chosen as host for these experiments. In previous studiesTrichoderma EGII (endoglucanase II) enzyme has been shown to causedetrimental effects to cotton fibre structures and thus to weaken thestrength properties of cotton-containing fabrics (In: Miettinen-Oinonenet al.: Effects of cellulases on cotton fiber and fabrics. In:Proceedings of the TIWC96 Conference, 1996, Vol. 1 (2), pp. 197.).

[0355] Stone-washing experiments were performed at pH 6.5 and 7 asdescribed in Example 3 except that no Berol was used.

[0356] The tested Trichoderma cellulase preparations were:

[0357] ALKO3133 (egl2 and cbh2 deleted)

[0358] ALKO3269 (egl2 and egl1 deleted)

[0359] ALKO3268 (egl2 and cbh1 deleted)

[0360] The dosage of Trichoderma preparations was about 2.5 mg (=lowdosage, L) or about 5 mg (=high dosage, H) of total protein per g offabric. 0.4 mg of purified 20K-cellulase per g of fabric was used whenneeded.

[0361] Results of color measurements of treated denim fabrics are shownin Table XIX.

[0362] The stone-washing results show that ALKO3269 (egl2 and egl1deleted) background causes less backstaining at neutral conditions thanALKO3268 (egl2 and cbh1 deleted) or ALKO3133 (egl2 and cbh2 deleted)background. Thus the preferred host for 20K-cellulase production forbiostoning is an ALKO3269-like strain. Although with higher20K-cellulase concentrations the Trichoderma background has probablyonly very minor importance. An ALKO3269-like background is probably asgood for 50K-cellulase and 50K-cellulase B production for biostoning asit is for 20K-cellulase production. TABLE XIX Color measurements ofdenim fabrics treated with different Trichoderma cellulase preparationswith (+) or without (−) 20K-cellulase. preparation/ 20K Right sideReverse side dosage +/− pH L b deltaE L b deltaE — − 6.5 2.2 1.1 3.1 0.70.1 1.4 ALKO3620/L − 6.5 2.2 2.6 3.0 −0.7 2.6 2.9 ALKO3620/L + 6.5 5.54.0 7.7 −1.3 5.0 5.5 ALKO3133/L − 6.5 1.9 2.2 3.7 0.2 1.6 2.3 ALKO3133/H− 6.5 4.2 1.9 4.5 −1.5 3.3 4.8 ALKO3133/L + 6.5 5.7 4.3 7.8 0.3 4.5 5.0ALKO3133/H + 6.5 8.5 4.0 9.4 −1.4 5.9 7.8 ALKO3269/L − 6.5 2.9 1.9 4.40.8 0.8 1.6 ALKO3269/H − 6.5 4.3 1.5 4.5 0.6 1.3 2.6 ALKO3269/L + 6.56.6 4.2 8.7 1.1 4.0 4.3 ALKO3269/H + 6.5 7.9 3.9 8.5 0.7 3.7 5.1ALKO3268/L − 6.5 2.9 1.7 3.7 0.1 1.8 3.0 ALKO3268/H − 6.5 4.2 2.0 4.3−0.7 3.4 5.0 ALKO3268/L + 6.5 5.9 3.2 7.7 −1.2 4.5 6.0 ALKO3268/H + 6.57.1 3.7 7.7 −2.0 5.8 7.3 — − 7.0 2.9 0.8 2.6 0.7 0.5 1.5 ALKO3620/L −7.0 3.3 1.2 1.9 1.7 0.3 1.1 ALKO3620/L + 7.0 6.7 3.4 5.6 1.1 3.2 2.9ALKO3133/L − 7.0 3.2 1.0 1.4 0.6 0.6 0.9 ALKO3133/L + 7.0 5.9 3.7 5.50.1 4.3 3.1 ALKO3269/L − 7.0 3.6 1.2 2.2 1.3 −0.3 1.3 ALKO3269/L + 7.06.4 3.4 5.9 1.2 3.2 2.8 ALKO3268/L − 7.0 2.9 1.4 3.9 0.5 0.4 2.5ALKO3268/L + 7.0 8.4 3.1 9.6 1.1 3.5 4.6

Example 29 Production of Melanocarpus albomyces ALKO4237 20K-Cellulasein T. reesei

[0363] The Trichoderma reesei strains were constructed for Melanocarpusalbomyces ALKO4237 20K-cellulase production. Strains produceMelanocarpus 20K-cellulase and are unable to produce T. reesei'sendoglucanase II and cellobiohydrolase I or endoglucanase I. Suchpreparations deficient in Trichoderma cellulolytic activity, and themaking of same by recombinant DNA methods, are described in U.S. Pat.No. 5,298,405 or Suominen et al. (1993) High frequency one-step genereplacement in Trichoderma reesei. II. Effects of deletions ofindividual cellulase genes. Mol. Gen. Genet. 241: 523., incorporatedherein by reference.

[0364] In construction of the Melanocarpus albomyces 20K-cellulaseproducing strains, the parental Trichoderma reesei strain ALKO3620 wastransformed with the expression cassettes from the plasmid pALK1231 orpALK1235 (FIGS. 28 and 29). In the cassettes 20K-cellulase is expressedfrom the strong cbh1 promoter. The integration of the expressioncassettes resulted in the replacements of the parental cbh1 (pALK1231)or the egl1 (pALK1235) genes.

[0365] In the host strain ALKO3620 the egl2 gene has been replaced bythe 3.3 kb XbaI-BglII fragment of the ble gene from Streptoalloteichushindustanus (Mattern et al. (1988) A vector of Aspergillustransformation conferring phleomycin resistance. Fungal Genet Newslett.35: 25.; Drocourt et al. (1990) Cassettes of the Streptoalloteichushindustanus ble gene for transformation of lower and higher eukaryotesto phleomycin resistance. Nucl. Acids Res. 18: 4009.) using therecombinant DNA methods described in U.S. Pat. No. 5,298,405,incorporated herein by reference.

[0366] The plasmids pALK1231 and pALK1235 that were used in theconstruction of the Melanocarpus cellulase producing strains areidentical to each other with respect to cbh1 promoter, 20K-cellulasegene and cbh1 terminator which are described below:

[0367]T. reesei cbh1 (cellobiohydrolase 1) promoter: The promoter isfrom Trichoderma reesei VTT-D-80133 (Teeri et al. (1983) The molecularcloning of the major cellulase gene from Trichoderma reesei.Bio/Technology 1: 696.). The 2.2 kb EcoRI-SacII fragment (Karhunen etal. (1993) High frequency one-step gene replacement in Trichodermareesei. I. Endoglucanase I overproduction. Mol Gen. Genet. 241: 515.)was used in the construct. The sequence of the promoter area preceedingthe ATG was published by Shoemaker et al. (1983) Molecular cloning ofexo-cellobiohydrolase from Trichoderma reesei strain L27.Bio/Technology 1. 691.). The last 15 nucleotides of the T reesei L27cbh1 promoter (the SacII site is underlined) are CCGCGGACTGGCATC(Shoemaker et al. 1983). The cbh1 promoter from the T reesei strainVTT-D-80133 has been sequenced at Alko Research Laboratories, and an onenucleotide difference in the DNA sequence has been noticed within theabove mentioned region. In the T reesei strain VTT-D-80133 the sequencepreceeding the ATG is CCGCGGACTG/C/GCATC (the SacII site is underlined,the additional cytosine in the DNA sequence is between the slashes).

[0368] The nucleotides missing from the promoter (10 bps after the SacIIto the ATG) were added and the exact promoter fusion to the first ATG ofthe Melanocarpus 20K-cellulase (see below) was done by using the PCR(polymerase chain reaction) method. The fusion and the PCR fragment weresequenced to ensure that no errors had occurred in the reaction. InpALK1231 the promoter area is also functioning as a homologous DNA(together with the cbh1 3′-fragment; see below) to target theintegration of the transforming DNA into the cbh1 locus.

[0369]Melanocarpus albomyces 20K-cellulase gene: The nucleotide sequenceand deduced amino acid sequence of the 20K-cellulase gene encoding an 20kDa cellulase is presented in Example 20 (FIG. 19). A 0.9 kb fragmentbeginning from ATG-codon was used in both plasmids.

[0370]T. reesei cbh1 terminator: The 739 bp AvaII fragment (Karhunen etal. (1993) High frequency one-step gene replacement in Trichodermareesei. I. Endoglucanase I overproduction. Mol. Gen. Genet. 241: 515.)starting 113 bp before the STOP codon of the cbh1 gene was added afterthe 20K-cellulase gene to ensure termination of transcription.

[0371] In addition the material described above the plasmid pALK1231contains:

[0372] amdS gene: The gene has been isolated from Aspergillus nidulansVH1-TRSX6 and it is coding for acetamidase (Hynes et al. (1983)Isolation of genomic clones containing the amdS gene of Aspergillusnidulans and their use in the analysis of the structural and regulatorymutations. Mol. Cell. Biol. 3: 1430.). Acetamidase enables the strain togrow by using acetamide as the only nitrogen source and thischaracteristics has been used for selecting the transformants. The 3.1kb fragment (SpeI-XbaI) from the plasmid p3SR2 (Kelly J. and Hynes M.(1985) Transformation of Aspergillus niger by the amdS gene ofAspergillus nidulans. EMBO J. 4: 475.) is used in the plasmids. Thefragment contains 1007 bps of the promoter area, 1897 bps of the codingregion (introns included) and the 183 bps terminator area of the amdSgene.

[0373] cbh1 3′-fragment: The fragment was isolated from T. reeseiALKO2466 by using plasmid rescue (1.7 kb, BamHI-EcoRI, starting 1.4 kbafter the gene's STOP, Suominen et al. (1993) High frequency one-stepgene replacement in Trichoderma reesei. II. Effects of deletions ofindividual cellulase genes. Mol. Gen. Genet. 241: 523.). Strain ALKO2466derives from the strain ALKO233 (Harkki et al. (1991) Geneticengineering of Trichoderma to produce strains with novel cellulaseprofiles. Enzyme Microb. Technol. 13: 227.). 3′-fragment is usedtogether with the promoter area to target the 20K-cellulase gene to thecbh1 locus by homologous recombination.

[0374] The plasmid pALK1235 contains:

[0375] hph gene: The gene encoding HmB phosphotransferase is originallyisolated from E. coli K-12 JM109 (Yanish-Perron et al. (1985) ImprovedM13 phage cloning vectors and host strains: nucleotide sequences of theM13mp18 and pUC19 vectors. Gene 33: 103.) and it confers resistance tohygromycin B (HmB). Resistance to hygromycin (inactivated byphosphorylation by HmB phosphotransferase) was used for selecting thetransformants. The hph gene together with the pki promoter and cbh2terminator (see below) is isolated from plasmid pRLM_(ex)30 (Mach et al.(1994) Transformation of Trichoderma reesei based on hygromycin Bresistance using homologous expression signals. Curr. Genet. 25: 567.)as a 2.2 kb NotI-PvuII fragment.

[0376] pki promoter: The about 0.75 kb pki (pyruvate kinase) promoterfor expressing hph has been synthesized by PCR using T. reesei QM 9414DNA as a template (Schindler et al. (1993) Characterization of thepyruvate kinase-encoding gene (pki1) of Trichoderma reesei. Gene 130:271.).

[0377] cbh2 terminator: The cbh2 terminator sequence starts immediatelyafter the STOP codon of the cbh2 gene (to the PvuII site 0.5 kb from theSTOP codon; Mach et al. (1994) Transformation of Trichoderma reeseibased on hygromycin B resistance using homologous expression signals.Curr. Genet. 25: 567.) and originates from plasmid pRLM_(ex)30.

[0378] egl1 5′-fragment: The 1.8 kb egl1 5′-fragment (ScaI-StuI) hasbeen isolated from T. reesei QM 6a (Mandels and Reese (1957) Inductionof cellulase in Trichoderma viridae as influenced by carbon sources andmetals. J. Bacteriol. 73: 269.). This fragment is situated about 1.35 kbupstream from the egl1 coding region and it was used to target theintegration of the the transforming DNA into the egl1 locus.

[0379] egl1 3′-fragment: The 1.6 kb egl1 3′-fragment (ScaI-XhoI) was,like the 5′-fragment, isolated from T. reesei QM 6a. The fragment issituated 0.3 kb downstream from the end of the egl1 gene and it was usedfor targeting of the transforming DNA into the egl1 locus.

[0380] The standard DNA methods described by Sambrook et al. (1989) In:Molecular cloning: a laboratory manual, 2nd ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. were used in construction ofthe vectors. The restriction enzymes, T4 DNA ligase, Klenow fragment ofthe DNA polymerase I, T4 DNA polymerase, polynucleotide kinase and Taqpolymerase were from Boehringer Mannheim, Germany) and New EnglandBiolabs (USA).

[0381] Each enzyme was used according to the supplier's instructions.Plasmid DNA was isolated by using Qiagen columns (Qiagen GmbH, Germany)or Promega Magic Minipreps (Promega, USA) according to themanufacturer's protocols. The oligonucleotides used in the PCR-reactionsand in sequencing reactions were synthetized by a ABI (AppliedBiosystems, USA) 381 A DNA Synthetizer. DNA sequencing was done asdescribed in Example 19.

[0382] DNA fragments for cloning or transformations were isolated fromlow-melting-point agarose gels (FMC Bioproducts, USA) by β-agarase Itreatment (New England Biolabs, USA) or by using the QIAEX GelExtraction Kit (Qiagen GmbH, Germany) according to the supplier'sinstructions.

[0383]T. reesei ALKO3620 was transformed as described by Penttilä et al.(1987) A versatile transformation system for the cellulolyticfilamentous fungus Trichoderma reesei. Gene 61: 155.) with themodifications described in Karhunen et al. (1993) High frequencyone-step gene replacement in Trichoderma reesei. I. Endoglucanase Ioverproduction. Mol. Gen. Genet. 241: 515.). T reesei transformants weretransferred on a selective medium and purified through conidia.Transformants were stabilized by growing them on selective slants fortwo generations prior to sporulating on potato dextrose agar.

Example 30 Characteristics of the Melanocarpus albomyces ALKO423720K-Cellulase Producing Transformants

[0384] The purified transformants were grown in shake flasks in a mediumcontaining 4% whey, 1.5% complex nitrogen source derived from grain, 5%KH₂PO₄ and 0.5% (NH₄)₂SO₄. Cultures were grown at 30° C. and 250 rpm for7 days.

[0385] The culture supernatants were blotted directly ontonitrocellulose filters by a dot-blot apparatus. CBHI was detected byimmunostaining using a CBHI specific monoclonal antibody CI-258 and EGIby spesific monoclonal antibody EI-2 (Aho et al. (1991) Monoclonalantibodies against core and cellulose-binding domains of Trichodermareesei cellobiohydrolases I and II and endoglucanase 1. Eur. J. Biochem.200: 643.) and the ProtoBlot Western blot AP system (Promega. USA)according to the recommendations of the manufacturer.

[0386] The T. reesei strains ALKO3620/pALK1231/14, ALKO3620/pALK1231/16,ALKO3620/pALK1231/20 and ALKO3620/pALK1231/59 do not contain the cbh1gene. The cbh1 gene is replaced by the amdS marker gene and the20K-cellulase construct in pALK1231 expression cassette. The cbh1 genereplacement was verified in Southern hybridisations. The T reeseistrains ALKO3620/pALK1235/40 and ALKO3620/pALK1235/49 do not contain theegl1 gene. The egl1 gene is replaced by the hph marker gene and the20K-cellulase construct in pALK1235 expression cassettes. The egl1 genereplacement was verified in Southern hybridisations. The host strainALKO3620 used in the transformations is deficient of the egl2 gene(replaced by ble gene from Streptoalloteichus hindustanus (Mattern etal., 1988, Drocourt et al., 1990). Thus the strains do not produceTrichoderma's cellulase components EGII and CBHI or EGI.

[0387] Samples from the culture supernatants were run on polyacrylamideslab gels containing 0.1% SDS on Bio-Rad Mini Protean II electrophoresissystem (USA). The polyclonal antibody prepared against the purified20K-cellulase was used to detect the produced protein in Western blots.In the detection, Promega's ProtoBlot® AP System was used. The Westernresult is shown in FIG. 30. The transformants ALKO3620/pALK1235/49,ALKO3620/pALK1235/40, ALKO3620/pALK1231/14 and ALKO3620/pALK1231/16(lanes 1, 2, 4 and 5) produce a protein which reacts with the polyclonal20K-cellulase antiserum. The size of the protein produced bytransformants is same as the size of purified 20K-cellulase (lane 6).ALKO3620 (lane 3) does not produce corresponding protein.

[0388] The endoglucanase activities of the transformants were determinedas described in Example 10. When 2% carboxymethylcellulose (CMC) wasused as a substrate reaction temperature was lifted up to 70° C. andthus the endoglucanase activity of ALKO3620 was heat inactivated. Whenusing 1% hydroxyethylcellulose as a substrate heat inactivation wasperformed before enzymatic activity measurements. Samples from growthmedium were diluted to 0.05 M HEPES, pH 7.0-buffer and incubated 20 minin 70° C. Heat inactivation of endoglucanase I (the major endoglucanaseleft in ALKO3620) was almost complete. The activity of egl1-negativetransformants dropped about 30% in heat inactivation which indicates theminor heat inactivation of 20K-cellulase. The endoglucanase activitiesare presented in Table XX. When HEC was the substrate, the 20K-cellulaseactivity was extrapolated to the activity before the heat treatment bydividing the activity obtained after the heat treatment with 0.7. TABLEXX The endoglucanase activities of T. reesei transformants producingMelanocarpus albomyces 20K-cellulase. 20K-cellulase activity (artificialunits/ml) CMC HEC Substrate 70° C., pH 7.0 50° C., pH 7.0 ALKO4237 —* 100** ALKO3620    50***   38*** ALKO3620/pALK1231/14 2400 350ALKO3620/pALK1231/16 2600 350 ALKO3620/pALK1231/20 6500 750ALKO3620/pALK1231/59 6800 750 ALKO3620/pALK1235/40 2400 325ALKO3620/pALK1235/49 2100 350

[0389] The endoglucanase activities of the T. reesei host strainALKO3620 are almost totally heat inactivated at 70° C. Melanocarpusalbomyces 20K-cellulase producing transformants produce substantialamounts of relative heat stable 20K-cellulase. The endoglucanaseproduction level of transformants is several times higher than that of20K-cellulase parental strain ALKO4237.

Example 31

[0390] Production of Melanocarpus albomyces ALKO4237 50K-Cellulase in T.reesei

[0391] The Trichoderma reesei strains were constructed for Melanocarpusalbomyces ALKO4237 50K-cellulase production. Strains produceMelanocarpus 50K-cellulase and are unable to produce T. reesei'sendoglucanase II and cellobiohydrolase I or endoglucanase I. Inconstruction of the Melanocarpus albomyces 50K-cellulase producingstrains, the parental Trichoderma reesei strain ALKO3620 was transformedwith the expression cassettes from the plasmid pALK1238 or pALK1240(FIGS. 31 and 32). In the cassettes 50K-cellulase is expressed from thestrong cbh1 promoter. The integration of the expression cassettesresults in the replacements of the parental cbh1 (pALK1238) or the egl1(pALK1240) genes. Cloning and transformation were done as described inExample 29, except that 20K-cellulase gene was replaced by 50K-cellulasegene (1.7 kb fragment beginning from ATG-codon) described in Example 22.The Melanocarpus albomyces 50K-cellulase producing transformants arethen characterized similar to example 30 with modifications obvious to aperson skilled in the art. The Melanocarpus albomyces 50K-cellulase Band protein-with-CBD producing transformants can be created similar toExamples 29 and 30 with modifications obvious to a person skilled in theart.

[0392] Having now fully described the invention, it will be understoodby those with skill in the art that the invention may be performedwithin a wide and equivalent range of conditions, parameters and thelike, without affecting the spirit or scope of the invention or anyembodiment thereof. All references cited herein are fully incorporatedherein by reference.

1. A nucleic acid molecule encoding a polypeptide having the enzymaticactivity of a cellulase, selected from the group consisting of: (a)nucleic acid molecules encoding a polypeptide comprising the amino acidsequence as depicted in FIG. 19 or 21; (b) nucleic acid moleculesencoding a polypeptide comprising the amino acid sequence as depicted inFIG. 23 or 27; (c) nucleic acid molecules comprising the coding sequenceof the nucleotide sequence as depicted in —FIG. 19 or 21; (d) nucleicacid molecules comprising the coding sequence of the nucleotide sequenceas depicted in FIG. 23 or 27; (e) nucleic acid molecules encoding apolypeptide comprising the amino acid sequence encoded by the DNA insertcontained in DSM 11024, DSM 11012, DSM 11025 or DSM 11014; (f) nucleicacid molecules encoding a polypeptide comprising the amino acid sequenceencoded by the DNA insert contained in DSM 11026, DSM 11011, DSM 11013or DSM 11027; (g) nucleic acid molecules comprising the coding sequenceof the DNA insert contained in DSM 11024, DSM 11012, DSM 11025 or DSM11014; (h) nucleic acid molecules comprising the coding sequence of theDNA insert contained in DSM 11026, DSM 11011, DSM 11013 or DSM 11027;(i) nucleic acid molecules hybridizing to a molecule of any one of (a),(c), (e) or (g); and (j) nucleic acid molecules the coding sequence ofwhich differs from the coding sequence of a nucleic acid molecule of anyone of (a) to (i) due to the degeneracy of the genetic code: (k) nucleicacid molecules encoding a polypeptide having cellulase activity andhaving an amino acid sequence which shows at least 80% identity to asequence as depicted in FIGS. 19, 21, 23 or
 27. 2. The nucleic acidmolecule of claim 1 which is RNA.
 3. The nucleic acid molecule of claim1 which is DNA.
 4. The DNA of claim 3 which is genomic DNA or cDNA.
 5. Avector containing a nucleic acid molecule of any one of claims 1 to 4.6. The vector of claim 5, in which the nucleic acid molecule is operablylinked to expression control sequences allowing expression inprokaryotic or eukaryotic host cells.
 7. A host cell transformed with anucleic acid molecule of any one of claims 1 to 4 or with a vector ofclaim 5 or
 6. 8. The host cell of claim 7 which belongs to filamentousfingi.
 9. The host cell of claims 7 to 8 which belongs to the genusTrichoderma or Aspergillus.
 10. The host cell of claim 9 which isTrichoderma reesei.
 11. A process for the production of a polypeptidehaving cellulase activity comprising the steps of culturing the hostcell of any one of claims 7 to 10 and recovering the protein from theculture medium.
 12. A polypeptide having cellulase activity encoded by anucleic acid molecule of any one of claims 1 to 4, a vector of claim 5or 6 and obtainable by the process of claim
 11. 13. An antibodyspecifically recognizing the polypeptide of claim
 12. 14. Anoligonucleotide specifically hybridizing to a nucleic acid molecule ofany one of claims 1 to
 4. 15. A process for the preparation of an enzymepreparation comprising a polypeptide of claim 12 comprising the steps ofculturing a host cell of any one of claims 7 to 10 and either recoveringthe polypeptide from the cells or separating the cells from the culturemedium and obtaining the supernatant.
 16. An enzyme preparationobtainable by the process of claim
 15. 17. An enzyme preparationcomprising at least one cellulase of a fungal species belonging to afungal genus selected from the group consisting of Melanocarpus,Myriococcum, Sporotrichum, Myceliophthora or Chaetomium.
 18. The enzymepreparation of claim 17, wherein the fungal species is Melanocarpusalbomyces, Myriococcum albomyces, Myriococcum sp. species represented byCBS 687.95, Sporotrichum thermophile, Myceliophthora thermophila orChaetomium thermophilum.
 19. The enzyme preparation of claim 17 or 18,wherein the fungus is Melanocarpus albomyces or Myriococcum albomycesCBS 685.95, Myriococcum sp. CBS 687.95, Sporotrichum thermophile CBS688.95 or Myceliophthora thermophila CBS 689.95 or Chaetomiumthermophilum CBS 730.95.
 20. The enzyme preparation of claims 16 to 19,which is liquid.
 21. The enzyme preparation of any one of claims 16 to19, which is dry.
 22. A method for biostoning which comprises the stepof adding the preparation of any one of claims 16 to 19 to cottoncontaining fabric or garments.
 23. The method of claim 22, wherein thefabric or garments is denim.
 24. A method for biofinishing, whichcomprises the step of adding the preparation of any one of claims 16 to19 to textile materials like fabrics or garments or yarn.
 25. The methodof claim 24, wherein the textile materials are manufactured of naturalcellulose containing fibers or manmade cellulose containing fibers orare mixtures thereof.
 26. The method of claim 24, wherein the textilematerials are blends of synthetic fibers and cellulose containingfibers.
 27. A detergent composition comprising the enzyme preparation ofclaims 16 to 19 and a surface active agent or surfactant.
 28. A methodof treating cellulosic fiber containing textile material, wherein saidmethod comprises mixing said textile material with the detergentcomposition of claim
 27. 29. A method for treating wood-derived pulp orfiber, which comprises the step of adding the enzyme preparation of anyone of claims 16 to 19 to wood-derived mechanical or chemical pulp orsecondary fiber.
 30. A method for improving the quality of animal feed,which comprises treating plant material with the enzyme preparation ofany one of claims 16 to 19.